Isolated human enzyme proteins, nucleic acid molecules encoding human enzyme proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the enzyme peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the enzyme peptides, and methods of identifying modulators of the enzyme peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of enzyme proteins that arerelated to the sulfatase enzyme subfamily, recombinant DNA molecules,and protein production. The present invention specifically providesnovel peptides and proteins that effect protein phosphorylation andnucleic acid molecules encoding such peptide and protein molecules, allof which are useful in the development of human therapeutics anddiagnostic compositions and methods.

BACKGROUND OF THE INVENTION

[0002] Many human enzymes serve as targets for the action ofpharmaceutically active compounds. Several classes of human enzymes thatserve as such targets include helicase, steroid esterase and sulfatase,convertase, synthase, dehydrogenase, monoxygenase, transferase, kinase,glutanase, decarboxylase, isomerase and reductase. It is thereforeimportant in developing new pharmaceutical compounds to identify targetenzyme proteins that can be put into high-throughput screening formats.The present invention advances the state of the art by providing novelhuman drug target enzymes related to the sulfatase subfamily.

Sulfatases

[0003] The novel human protein, and encoding gene, provided by thepresent invention is related to the sulfatase family of enzymes,including estrone sulfatases. Specifically, the novel human protein ofthe present invention is a novel alternative splice form of a geneprovided in Genbank gi5689491 and published PCT patent applicationWO200055629 (see the BLAST and Genewise alignments of the sequences ofthe present invention and the sequences of gi5689491 and WO200055629provided in FIG. 2). The evidence supporting alternative splicingincludes a different polyA signal used to create the protein of thepresent invention compared with the art-known protein; the stop codon atcDNA positions 1223-1225 and polyA signal at cDNA positions 1750-1756are present in the genomic sequence; and the last exon of the cDNA ofthe present invention crosses the splicing site of the correspondingexon 5 of the art-known protein (these differences are illustrated inFIG. 2 in the BLAST and Genewise alignments of the sequences of thepresent invention and the sequences of gi5689491 and WO200055629).

[0004] Novel human sulfatases, such as the protein provided by presentinvention, are particularly useful as targets for treating cancer,particularly breast cancer. Sulfatases are important for generatingestrone and 5-androstenediol from sulfated precursors. As stated byPurohit et al., (Mol Cell Endocrinol Jan. 22, 2001 ;171(1-2):129-135),“The development of inhibitors to block the formation of estrone and5-androstenediol from sulfated precursors is an important new strategyfor the treatment of breast cancer”. Thus, sulfatase inhibitors areuseful for treating cancer and, consequently, novel sulfatase proteinsare valuable as novel targets for the development of anti-cancertherapeutic agents. Purohit et al. found that non-steroidal andsteroidal sulfamates, particularly a tricyclic coumarin sulfamate (“667COUMATE”) and 2-methoxyestrone-3-O-sulfamate (2-MeOEMATE), inhibitedestrone sulfatase activity and “offer considerable potential fordevelopment for cancer therapy”. The importance of sulfatases relatingin breast cancer is further described in published PCT patentapplication WO200055629, “Novel Methods of Diagnosing and TreatingBreast Cancer, Compositions, and Methods of Screening for Breast CancerModulators”.

[0005] Enzyme proteins, particularly members of the sulfatase enzymesubfamily, are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown members of thissubfamily of enzyme proteins. The present invention advances the stateof the art by providing previously unidentified human enzyme proteins,and the polynucleotides encoding them, that have homology to members ofthe sulfatase enzyme subfamily. These novel compositions are useful inthe diagnosis, prevention and treatment of biological processesassociated with human diseases.

SUMMARY OF THE INVENTION

[0006] The present invention is based in part on the identification ofamino acid sequences of human enzyme peptides and proteins that arerelated to the sulfatase enzyme subfamily, as well as allelic variantsand other mammalian orthologs thereof. These unique peptide sequences,and nucleic acid sequences that encode these peptides, can be used asmodels for the development of human therapeutic targets, aid in theidentification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate enzyme activity incells and tissues that express the enzyme. Experimental data as providedin FIG. 1 indicates expression in humans in pancreas adenocarcinoma,breast, colon, and brain.

DESCRIPTION OF THE FIGURE SHEETS

[0007]FIG. 1 provides the nucleotide sequence of a CDNA molecule thatencodes the enzyme protein of the present invention. (SEQ ID NO: 1) Inaddition, structure and functional information is provided, such as ATGstart, stop and tissue distribution, where available, that allows one toreadily determine specific uses of inventions based on this molecularsequence. Experimental data as provided in FIG. 1 indicates expressionin humans in pancreas adenocarcinoma, breast, colon, and brain.

[0008]FIG. 2 provides the predicted amino acid sequence of the enzyme ofthe present invention. (SEQ ID NO:2) In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

[0009]FIG. 3 provides genomic sequences that span the gene encoding theenzyme protein of the present invention. (SEQ ID NO:3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence.

DETAILED DESCRIPTION OF THE INVENTION General Description

[0010] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a enzyme protein or part of aenzyme protein and are related to the sulfatase enzyme subfamily.Utilizing these sequences, additional genomic sequences were assembledand transcript and/or cDNA sequences were isolated and characterized.Based on this analysis, the present invention provides amino acidsequences of human enzyme peptides and proteins that are related to thesulfatase enzyme subfamily, nucleic acid sequences in the form oftranscript sequences, cDNA sequences and/or genomic sequences thatencode these enzyme peptides and proteins, nucleic acid variation(allelic information), tissue distribution of expression, andinformation about the closest art known protein/peptide/domain that hasstructural or sequence homology to the enzyme of the present invention.

[0011] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known enzyme proteins of thesulfatase enzyme subfamily and the expression pattern observed.Experimental data as provided in FIG. 1 indicates expression in humansin pancreas adenocarcinoma, breast, colon, and brain. The art hasclearly established the commercial importance of members of this familyof proteins and proteins that have expression patterns similar to thatof the present gene. Some of the more specific features of the peptidesof the present invention, and the uses thereof, are described herein,particularly in the Background of the Invention and in the annotationprovided in the Figures, and/or are known within the art for each of theknown sulfatase family or subfamily of enzyme proteins.

SPECIFIC EMBODIMENTS Peptide Molecules

[0012] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of theenzyme family of proteins and are related to the sulfatase enzymesubfamily (protein sequences are provided in FIG. 2, transcript/cDNAsequences are provided in FIG. 1 and genomic sequences are provided inFIG. 3). The peptide sequences provided in FIG. 2, as well as theobvious variants described herein, particularly allelic variants asidentified herein and using the information in FIG. 3, will be referredherein as the enzyme peptides of the present invention, enzyme peptides,or peptides/proteins of the present invention.

[0013] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprise the aminoacid sequences of the enzyme peptides disclosed in the FIG. 2, (encodedby the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

[0014] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0015] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0016] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of theenzyme peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0017] The isolated enzyme peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inhumans in pancreas adenocarcinoma, breast, colon, and brain. Forexample, a nucleic acid molecule encoding the enzyme peptide is clonedinto an expression vector, the expression vector introduced into a hostcell and the protein expressed in the host cell. The protein can then beisolated from the cells by an appropriate purification scheme usingstandard protein purification techniques. Many of these techniques aredescribed in detail below.

[0018] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided inFIG. 3 (SEQ ID NO:3). The amino acid sequence of such a protein isprovided in FIG. 2. A protein consists of an amino acid sequence whenthe amino acid sequence is the final amino acid sequence of the protein.

[0019] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ IDNO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequencesprovided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0020] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the enzyme peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

[0021] The enzyme peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a enzyme peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the enzyme peptide. “Operatively linked”indicates that the enzyme peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the enzyme peptide.

[0022] In some uses, the fusion protein does not affect the activity ofthe enzyme peptide per se. For example, the fusion protein can include,but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant enzyme peptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

[0023] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A enzyme peptide-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the enzyme peptide.

[0024] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0025] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the enzyme peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0026] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of a reference sequence is aligned forcomparison purposes. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0027] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version2.0), using a PAM120 weight residue table, a gap length penalty of 12and a gap penalty of 4.

[0028] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0029] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the enzyme peptides of the present invention as well as beingencoded by the same genetic locus as the enzyme peptide provided herein.The gene encoding the novel enzyme of the present invention is locatedon a genome component that has been mapped to human chromosome 8 (asindicated in FIG. 3), which is supported by multiple lines of evidence,such as STS and BAC map data.

[0030] Allelic variants of a enzyme peptide can readily be identified asbeing a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the enzyme peptide as well asbeing encoded by the same genetic locus as the enzyme peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in FIG. 3, such as the genomic sequence mapped tothe reference human. The gene encoding the novel enzyme of the presentinvention is located on a genome component that has been mapped to humanchromosome 8 (as indicated in FIG. 3), which is supported by multiplelines of evidence, such as STS and BAC map data. As used herein, twoproteins (or a region of the proteins) have significant homology whenthe amino acid sequences are typically at least about 70-80%, 80-90%,and more typically at least about 90-95% or more homologous. Asignificantly homologous amino acid sequence, according to the presentinvention, will be encoded by a nucleic acid sequence that willhybridize to a enzyme peptide encoding nucleic acid molecule understringent conditions as more fully described below.

[0031] Paralogs of a enzyme peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the enzyme peptide, as being encoded by a gene from hurnans,and as having similar activity or function. Two proteins will typicallybe considered paralogs when the amino acid sequences are typically atleast about 60% or greater, and more typically at least about 70% orgreater homology through a given region or domain. Such paralogs will beencoded by a nucleic acid sequence that will hybridize to a enzymepeptide encoding nucleic acid molecule under moderate to stringentconditions as more fully described below.

[0032] Orthologs of a enzyme peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the enzyme peptide as well as being encoded by a gene fromanother organism. Preferred orthologs will be isolated from mammals,preferably primates, for the development of human therapeutic targetsand agents. Such orthologs will be encoded by a nucleic acid sequencethat will hybridize to a enzyme peptide encoding nucleic acid moleculeunder moderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

[0033] Non-naturally occurring variants of the enzyme peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the enzyme peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a enzyme peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Tbr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal.,Science 247:1306-1310 (1990).

[0034] Variant enzyme peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

[0035] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0036] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al.,Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as enzyme activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0037] The present invention further provides fragments of the enzymepeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

[0038] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a enzyme peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the enzyme peptide or could be chosen forthe ability to perform a function, e.g. bind a substrate or act as animmunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe enzyme peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

[0039] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally inenzyme peptides are described in basic texts, detailed monographs, andthe research literature, and they are well known to those of skill inthe art (some of these features are identified in FIG. 2).

[0040] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0041] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y Acad Sci. 663:48-62(1992)).

[0042] Accordingly, the enzyme peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature enzyme peptide is fused withanother compound, such as a compound to increase the half-life of theenzyme peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature enzyme peptide, such as aleader or secretory sequence or a sequence for purification of themature enzyme peptide or a pro-protein sequence.

Protein/Peptide Uses

[0043] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a enzyme-effectorprotein interaction or enzyme-ligand interaction), the protein can beused to identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

[0044] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0045] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, enzymes isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the enzyme. Experimental data as providedin FIG. 1 indicates that the enzymes of the present invention areexpressed in humans in pancreas adenocarcinoma, breast, and colon, asindicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in fetal brain. A largepercentage of pharmaceutical agents are being developed that modulatethe activity of enzyme proteins, particularly members of the sulfatasesubfamily (see Background of the Invention). The structural andfunctional information provided in the Background and Figures providespecific and substantial uses for the molecules of the presentinvention, particularly in combination with the expression informationprovided in FIG. 1. Experimental data as provided in FIG. 1 indicatesexpression in humans in pancreas adenocarcinoma, breast, colon, andbrain. Such uses can readily be determined using the informationprovided herein, that which is known in the art, and routineexperimentation.

[0046] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to enzymes that are related tomembers of the sulfatase subfamily. Such assays involve any of the knownenzyme functions or activities or properties useful for diagnosis andtreatment of enzyme-related conditions that are specific for thesubfamily of enzymes that the one of the present invention belongs to,particularly in cells and tissues that express the enzyme. Experimentaldata as provided in FIG. 1 indicates that the enzymes of the presentinvention are expressed in humans in pancreas adenocarcinoma, breast,and colon, as indicated by virtual northern blot analysis. In addition,PCR-based tissue screening panels indicate expression in fetal brain.

[0047] The proteins of the present invention are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the enzyme, as a biopsyor expanded in cell culture. Experimental data as provided in FIG. 1indicates expression in humans in pancreas adenocarcinoma, breast,colon, and brain. In an alternate embodiment, cell-based assays involverecombinant host cells expressing the enzyme protein.

[0048] The polypeptides can be used to identify compounds that modulateenzyme activity of the protein in its natural state or an altered formthat causes a specific disease or pathology associated with the enzyme.Both the enzymes of the present invention and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the enzyme. These compounds can befurther screened against a functional enzyme to determine the effect ofthe compound on the enzyme activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the enzyme to a desired degree.

[0049] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the enzyme protein and a molecule that normally interacts withthe enzyme protein, e.g. a substrate or a component of the signalpathway that the enzyme protein normally interacts (for example, anotherenzyme). Such assays typically include the steps of combining the enzymeprotein with a candidate compound under conditions that allow the enzymeprotein, or fragment, to interact with the target molecule, and todetect the formation of a complex between the protein and the target orto detect the biochemical consequence of the interaction with the enzymeprotein and the target, such as any of the associated effects of signaltransduction such as protein phosphorylation, cAMP turnover, andadenylate cyclase activation, etc.

[0050] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D—and/or L—configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab ′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0051] One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantenzymes or appropriate fragments containing mutations that affect enzymefunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

[0052] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) enzyme activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate enzyme activity. Thus, the phosphorylation of asubstrate, activation of a protein, a change in the expression of genesthat are up-or down-regulated in response to the enzyme proteindependent signal cascade can be assayed.

[0053] Any of the biological or biochemical functions mediated by theenzyme can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the enzyme can be assayed.Experimental data as provided in FIG. 1 indicates that the enzymes ofthe present invention are expressed in humans in pancreasadenocarcinoma, breast, and colon, as indicated by virtual northern blotanalysis. In addition, PCR-based tissue screening panels indicateexpression in fetal brain.

[0054] Binding and/or activating compounds can also be screened by usingchimeric enzyme proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native enzyme. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the enzyme is derived.

[0055] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the enzyme (e.g. binding partners and/or ligands).Thus, a compound is exposed to a enzyme polypeptide under conditionsthat allow the compound to bind or to otherwise interact with thepolypeptide. Soluble enzyme polypeptide is also added to the mixture. Ifthe test compound interacts with the soluble enzyme polypeptide, itdecreases the amount of complex formed or activity from the enzymetarget. This type of assay is particularly useful in cases in whichcompounds are sought that interact with specific regions of the enzyme.Thus, the soluble polypeptide that competes with the target enzymeregion is designed to contain peptide sequences corresponding to theregion of interest.

[0056] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the enzyme protein, or fragment, or itstarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay.

[0057] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,MO) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of enzyme-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a enzyme-binding protein and a candidate compound are incubated inthe enzyme protein-presenting wells and the amount of complex trapped inthe well can be quantitated. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theenzyme protein target molecule, or which are reactive with enzymeprotein and compete with the target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with thetarget molecule.

[0058] Agents that modulate one of the enzymes of the present inventioncan be identified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

[0059] Modulators of enzyme protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the enzyme pathway, by treating cells or tissuesthat express the enzyme. Experimental data as provided in FIG. 1indicates expression in humans in pancreas adenocarcinoma, breast,colon, and brain. These methods of treatment include the steps ofadministering a modulator of enzyme activity in a pharmaceuticalcomposition to a subject in need of such treatment, the modulator beingidentified as described herein.

[0060] In yet another aspect of the invention, the enzyme proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the enzyme and are involved in enzyme activity.Such enzyme-binding proteins are also likely to be involved in thepropagation of signals by the enzyme proteins or enzyme targets as, forexample, downstream elements of a enzyme-mediated signaling pathway.Alternatively, such enzyme-binding proteins are likely to be enzymeinhibitors.

[0061] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a enzyme proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aenzyme-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the enzyme protein.

[0062] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a enzyme-modulating agent, an antisense enzymenucleic acid molecule, a enzyme-specific antibody, or a enzyme-bindingpartner) can be used in an animal or other model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal or other model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

[0063] The enzyme proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in humans in pancreas adenocarcinoma, breast,colon, and brain. The method involves contacting a biological samplewith a compound capable of interacting with the enzyme protein such thatthe interaction can be detected. Such an assay can be provided in asingle detection format or a multi-detection format such as an antibodychip array.

[0064] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0065] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered enzyme activity in cell-based orcell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

[0066] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0067] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.PharmacoL Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the enzyme protein in which one ormore of the enzyme functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and enzyme activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

[0068] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in pancreas adenocarcinoma, breast, colon, andbrain. Accordingly, methods for treatment include the use of the enzymeprotein or fragments.

Antibodies

[0069] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0070] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0071] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0072] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0073] Antibodies are preferably prepared from regions or discretefragments of the enzyme proteins. Antibodies can be prepared from anyregion of the peptide as described herein. However, preferred regionswill include those involved in function/activity and/or enzyme/bindingpartner interaction. FIG. 2 can be used to identify particularlyimportant regions while sequence alignment can be used to identifyconserved and unique sequence fragments.

[0074] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0075] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibody Uses

[0076] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Experimental data as provided in FIG. 1 indicatesthat the enzymes of the present invention are expressed in humans inpancreas adenocarcinoma, breast, and colon, as indicated by virtualnorthern blot analysis. In addition, PCR-based tissue screening panelsindicate expression in fetal brain. Further, such antibodies can be usedto detect protein in situ, in vitro, or in a cell lysate or supernatantin order to evaluate the abundance and pattern of expression. Also, suchantibodies can be used to assess abnormal tissue distribution orabnormal expression during development or progression of a biologicalcondition. Antibody detection of circulating fragments of the fulllength protein can be used to identify turnover.

[0077] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates expression in humans in pancreas adenocarcinoma, breast,colon, and brain. If a disorder is characterized by a specific mutationin the protein, antibodies specific for this mutant protein can be usedto assay for the presence of the specific mutant protein.

[0078] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin pancreas adenocarcinoma, breast, colon, and brain. The diagnosticuses can be applied, not only in genetic testing, but also in monitoringa treatment modality. Accordingly, where treatment is ultimately aimedat correcting expression level or the presence of aberrant sequence andaberrant tissue distribution or developmental expression, antibodiesdirected against the protein or relevant fragments can be used tomonitor therapeutic efficacy.

[0079] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0080] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in humans in pancreasadenocarcinoma, breast, colon, and brain. Thus, where a specific proteinhas been correlated with expression in a specific tissue, antibodiesthat are specific for this protein can be used to identify a tissuetype.

[0081] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the enzyme peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

[0082] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nuleic acid arrays and similar methods have been developed forantibody arrays.

Nucleic Acid Molecules

[0083] The present invention further provides isolated nucleic acidmolecules that encode a enzyme peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the enzyme peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0084] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated”nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5KB, 4KB,3KB, 2KB, or IKB or less, particularly contiguous peptide encodingsequences and peptide encoding sequences within the same gene butseparated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0085] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0086] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified partially or substantially) DNA molecules in solution. IsolatedRNA molecules include in vivo or in vitro RNA transcripts of theisolated DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

[0087] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO: 1, transcript sequence and SEQ ID NO:3, genomic sequence),or any nucleic acid molecule that encodes the protein provided in FIG.2, SEQ ID NO:2. A nucleic acid molecule consists of a nucleotidesequence when the nucleotide sequence is the complete nucleotidesequence of the nucleic acid molecule.

[0088] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

[0089] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0090] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0091] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0092] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the enzyme peptide alone,the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0093] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0094] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the enzyme proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0095] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0096] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0097] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0098] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. The gene encoding thenovel enzyme of the present invention is located on a genome componentthat has been mapped to human chromosome 8 (as indicated in FIG. 3),which is supported by multiple lines of evidence, such as STS and BACmap data.

[0099] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6X sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2 X SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

[0100] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.

[0101] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0102] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0103] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0104] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0105] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods. The gene encoding the novelenzyme of the present invention is located on a genome component thathas been mapped to human chromosome 8 (as indicated in FIG. 3), which issupported by multiple lines of evidence, such as STS and BAC map data.

[0106] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0107] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0108] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0109] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0110] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0111] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that the enzymes of the present invention are expressed inhumans in pancreas adenocarcinoma, breast, and colon, as indicated byvirtual northern blot analysis. In addition, PCR-based tissue screeningpanels indicate expression in fetal brain. Accordingly, the probes canbe used to detect the presence of, or to determine levels of, a specificnucleic acid molecule in cells, tissues, and in organisms. The nucleicacid whose level is determined can be DNA or RNA. Accordingly, probescorresponding to the peptides described herein can be used to assessexpression and/or gene copy number in a given cell, tissue, or organism.These uses are relevant for diagnosis of disorders involving an increaseor decrease in enzyme protein expression relative to normal results.

[0112] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

[0113] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a enzyme protein, such as bymeasuring a level of a enzyme-encoding nucleic acid in a sample of cellsfrom a subject e.g., mRNA or genomic DNA, or determining if a enzymegene has been mutated. Experimental data as provided in FIG. 1 indicatesthat the enzymes of the present invention are expressed in humans inpancreas adenocarcinoma, breast, and colon, as indicated by virtualnorthern blot analysis. In addition, PCR-based tissue screening panelsindicate expression in fetal brain.

[0114] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate enzyme nucleic acid expression.

[0115] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the enzyme gene, particularly biological and pathologicalprocesses that are mediated by the enzyme in cells and tissues thatexpress it. Experimental data as provided in FIG. 1 indicates expressionin humans in pancreas adenocarcinoma, breast, colon, and brain. Themethod typically includes assaying the ability of the compound tomodulate the expression of the enzyme nucleic acid and thus identifyinga compound that can be used to treat a disorder characterized byundesired enzyme nucleic acid expression. The assays can be performed incell-based and cell-free systems. Cell-based assays include cellsnaturally expressing the enzyme nucleic acid or recombinant cellsgenetically engineered to express specific nucleic acid sequences.

[0116] The assay for enzyme nucleic acid expression can involve directassay of nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in the signal pathway. Further, the expression ofgenes that are up-or down-regulated in response to the enzyme proteinsignal pathway can also be assayed. In this embodiment the regulatoryregions of these genes can be operably linked to a reporter gene such asluciferase.

[0117] Thus, modulators of enzyme gene expression can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of mRNA determined.

[0118] The level of expression of enzyme mRNA in the presence of thecandidate compound is compared to the level of expression of enzyme mRNAin the absence of the candidate compound. The candidate compound canthen be identified as a modulator of nucleic acid expression based onthis comparison and be used, for example to treat a disordercharacterized by aberrant nucleic acid expression. When expression ofmRNA is statistically significantly greater in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of nucleic acid expression. When nucleic acidexpression is statistically significantly less in the presence of thecandidate compound than in its absence, the candidate compound isidentified as an inhibitor of nucleic acid expression.

[0119] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate enzyme nucleic acid expressionin cells and tissues that express the enzyme. Experimental data asprovided in FIG. 1 indicates that the enzymes of the present inventionare expressed in humans in pancreas adenocarcinoma, breast, and colon,as indicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in fetal brain. Modulationincludes both up-regulation (i.e. activation or agonization) ordown-regulation (suppression or antagonization) or nucleic acidexpression.

[0120] Alternatively, a modulator for enzyme nucleic acid expression canbe a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits theenzyme nucleic acid expression in the cells and tissues that express theprotein. Experimental data as provided in FIG. 1 indicates expression inhumans in pancreas adenocarcinoma, breast, colon, and brain.

[0121] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe enzyme gene in clinical trials or in a treatment regimen. Thus, thegene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

[0122] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in enzyme nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in enzyme genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the enzyme gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the enzyme gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a enzyme protein.

[0123] Individuals carrying mutations in the enzyme gene can be detectedat the nucleic acid level by a variety of techniques. The gene encodingthe novel enzyme of the present invention is located on a genomecomponent that has been mapped to human chromosome 8 (as indicated inFIG. 3), which is supported by multiple lines of evidence, such as STSand BAC map data. Genomic DNA can be analyzed directly or can beamplified by using PCR prior to analysis. RNA or cDNA can be used in thesame way. In some uses, detection of the mutation involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can be particularly useful fordetecting point mutations in the gene (see Abravaya et al., NucleicAcids Res. 23:675-682 (1995)). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

[0124] Alternatively, mutations in a enzyme gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0125] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0126] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant enzyme gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0127] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.EnzymoL 217:286-295 (1992)), electrophoretic mobility of mutant and wildtype nucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cottonet al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

[0128] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the enzyme gene in an individual in order to select anappropriate compound or dosage regimen for treatment.

[0129] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0130] The nucleic acid molecules are thus useful as antisenseconstructs to control enzyme gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of enzyme protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of mRNA into enzyme protein.

[0131] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of enzyme nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired enzyme nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the MRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the enzyme protein, such as substratebinding.

[0132] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in enzyme geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desired enzymeprotein to treat the individual.

[0133] The invention also encompasses kits for detecting the presence ofa enzyme nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that the enzymes of the present inventionare expressed in humans in pancreas adenocarcinoma, breast, and colon,as indicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in fetal brain. For example,the kit can comprise reagents such as a labeled or labelable nucleicacid or agent capable of detecting enzyme nucleic acid in a biologicalsample; means for determining the amount of enzyme nucleic acid in thesample; and means for comparing the amount of enzyme nucleic acid in thesample with a standard. The compound or agent can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect enzyme protein mRNA or DNA.

Nucleic Acid Arrays

[0134] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ IDNOS:1 and 3).

[0135] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0136] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0137] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0138] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0139] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and CDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0140] Using such arrays, the present invention provides methods toidentify the expression of the enzyme proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of the enzymegene of the present invention.

[0141] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1 982), Vol.2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0142] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0143] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0144] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0145] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified enzyme gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

Vectors/host cells

[0146] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0147] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0148] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in prokaryotic or eukaryoticcells or in both (shuttle vectors).

[0149] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0150] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from Ecoli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0151] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0152] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0153] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0154] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0155] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0156] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0157] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enteroenzyme. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. Examplesof suitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301 -315 (1988)) and pET 11d (Studier et al.,Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).

[0158] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990)119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0159] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec I (Baldari, et al., EMBO J6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0160] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

[0161] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

[0162] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0163] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0164] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0165] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0166] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0167] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0168] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0169] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0170] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such asenzymes, appropriate secretion signals are incorporated into the vector.The signal sequence can be endogenous to the peptides or heterologous tothese peptides.

[0171] Where the peptide is not secreted into the medium, which istypically the case with enzymes, the protein can be isolated from thehost cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0172] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

Uses of vectors and host cells

[0173] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga enzyme protein or peptide that can be further purified to producedesired amounts of enzyme protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0174] Host cells are also useful for conducting cell-based assaysinvolving the enzyme protein or enzyme protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a native enzyme protein is useful forassaying compounds that stimulate or inhibit enzyme protein function.

[0175] Host cells are also useful for identifying enzyme protein mutantsin which these functions are affected. If the mutants naturally occurand give rise to a pathology, host cells containing the mutations areuseful to assay compounds that have a desired effect on the mutantenzyme protein (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native enzyme protein.

[0176] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a enzyme proteinand identifying and evaluating modulators of enzyme protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

[0177] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the enzyme proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0178] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the enzyme protein to particularcells.

[0179] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0180] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0181] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0182] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, enzyme protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo enzymeprotein function, including substrate interaction, the effect ofspecific mutant enzyme proteins on enzyme protein function and substrateinteraction, and the effect of chimeric enzyme proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more enzyme proteinfunctions.

[0183] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 6 1 1799 DNA Human 1 tatttcattt tagtctcacc gtctccgttt ttctctgactgcccagaact ccagaaatca 60 ggagacggag acattttgtc agttttgcaa cattggaccaaatacaatga agtattcttg 120 ctgtgctctg gttttggctg tcctgggcac agaattgctgggaagcctct gttcgactgt 180 cagatccccg aggttcagag gacggataca gcaggaacgaaaaaacatcc gacccaacat 240 tattcttgtg cttaccgatg atcaagatgt ggagctggggtccctgcaag tcatgaacaa 300 aacgagaaag attatggaac atgggggggc caccttcatcaatgcctttg tgactacacc 360 catgtgctgc ccgtcacggt cctccatgct caccgggaagtatgtgcaca atcacaatgt 420 ctacaccaac aacgagaact gctcttcccc ctcgtggcaggccatgcatg agcctcggac 480 ttttgctgta tatcttaaca acactggcta cagaacagccttttttggaa aatacctcaa 540 tgaatataat ggcagctaca tcccccctgg gtggcgagaatggcttggat taatcaagaa 600 ttctcgcttc tataattaca ctgtttgtcg caatggcatcaaagaaaagc atggatttga 660 ttatgcaaag gactacttca cagacttaat cactaacgagagcattaatt acttcaaaat 720 gtctaagaga atgtatcccc ataggcccgt tatgatggtgatcagccacg ctgcgcccca 780 cggccccgag gactcagccc cacagttttc taaactgtaccccaatgctt cccaacacat 840 aactcctagt tataactatg caccaaatat ggataaacactggattatgc agtacacagg 900 accaatgctg cccatccaca tggaatttac aaacattctacagcgcaaaa ggctccagac 960 tttgatgtca gtggatgatt ctgtggagag gctgtataacatgctcgtgg agacggggga 1020 gctggagaat acttacatca tttacaccgc cgaccatggttaccatattg ggcagtttgg 1080 actggtcaag gggaaatcca tgccatatga ctttgatattcgtgtgcctt tttttattcg 1140 tggtccaagt gtagaaccag gatcaatgta cgtatttctctgtttgcaac attcaactgt 1200 cgtacctcaa gtgtgtctaa gataattcaa ttaccagtctcagtatctgg tttcctttca 1260 tccaaaacaa aaaaggatgt gtgtaggctg gttaatttcgaagatgaaaa ccttttcctc 1320 cctgccacat cttaaattag ctcaagtata ctacttaaagagaaaggaaa aataagtgta 1380 tcaatgacta attctctcaa attgactgga atctatgtctttttggtctg tgtgcacaga 1440 caggatgtga tcttctggga tatcaccctt ctttgaatcagagatacgct gtcatttaaa 1500 aaaaaaacct gacaccatcc ttttagtgtt taacttttaaaaattattcc gaaagaaatg 1560 tttttaaaag ataaattttg aaaagctggc ttttcttttaaaggaaaaag agctaaagga 1620 ctaggctgct atttctgtca ctgtaggcag gtcactgcttctctttgcat ctctattttc 1680 ccatcatgaa atggccttgc ctattttccc atcataaaatggccttgtca atcatctcag 1740 gatgttttga ataaaatggg attgcatcca tgaaagaaaaaaaaaaaaaa aaaaaaaaa 1799 2 372 PRT Human 2 Met Lys Tyr Ser Cys Cys AlaLeu Val Leu Ala Val Leu Gly Thr Glu 1 5 10 15 Leu Leu Gly Ser Leu CysSer Thr Val Arg Ser Pro Arg Phe Arg Gly 20 25 30 Arg Ile Gln Gln Glu ArgLys Asn Ile Arg Pro Asn Ile Ile Leu Val 35 40 45 Leu Thr Asp Asp Gln AspVal Glu Leu Gly Ser Leu Gln Val Met Asn 50 55 60 Lys Thr Arg Lys Ile MetGlu His Gly Gly Ala Thr Phe Ile Asn Ala 65 70 75 80 Phe Val Thr Thr ProMet Cys Cys Pro Ser Arg Ser Ser Met Leu Thr 85 90 95 Gly Lys Tyr Val HisAsn His Asn Val Tyr Thr Asn Asn Glu Asn Cys 100 105 110 Ser Ser Pro SerTrp Gln Ala Met His Glu Pro Arg Thr Phe Ala Val 115 120 125 Tyr Leu AsnAsn Thr Gly Tyr Arg Thr Ala Phe Phe Gly Lys Tyr Leu 130 135 140 Asn GluTyr Asn Gly Ser Tyr Ile Pro Pro Gly Trp Arg Glu Trp Leu 145 150 155 160Gly Leu Ile Lys Asn Ser Arg Phe Tyr Asn Tyr Thr Val Cys Arg Asn 165 170175 Gly Ile Lys Glu Lys His Gly Phe Asp Tyr Ala Lys Asp Tyr Phe Thr 180185 190 Asp Leu Ile Thr Asn Glu Ser Ile Asn Tyr Phe Lys Met Ser Lys Arg195 200 205 Met Tyr Pro His Arg Pro Val Met Met Val Ile Ser His Ala AlaPro 210 215 220 His Gly Pro Glu Asp Ser Ala Pro Gln Phe Ser Lys Leu TyrPro Asn 225 230 235 240 Ala Ser Gln His Ile Thr Pro Ser Tyr Asn Tyr AlaPro Asn Met Asp 245 250 255 Lys His Trp Ile Met Gln Tyr Thr Gly Pro MetLeu Pro Ile His Met 260 265 270 Glu Phe Thr Asn Ile Leu Gln Arg Lys ArgLeu Gln Thr Leu Met Ser 275 280 285 Val Asp Asp Ser Val Glu Arg Leu TyrAsn Met Leu Val Glu Thr Gly 290 295 300 Glu Leu Glu Asn Thr Tyr Ile IleTyr Thr Ala Asp His Gly Tyr His 305 310 315 320 Ile Gly Gln Phe Gly LeuVal Lys Gly Lys Ser Met Pro Tyr Asp Phe 325 330 335 Asp Ile Arg Val ProPhe Phe Ile Arg Gly Pro Ser Val Glu Pro Gly 340 345 350 Ser Met Tyr ValPhe Leu Cys Leu Gln His Ser Thr Val Val Pro Gln 355 360 365 Val Cys LeuArg 370 3 42571 DNA Human 3 gtatcaggtt tctcacgatt taaaacaaat gcacagaaaccaaacagtca gtgcagaata 60 attgcaggct ttcagtgttc agcatgtaca gcaatcactgtggaatcacc ctgcgttatt 120 aagaagaaag caccaaatct tacattagtg acttctacagggctgcgtta tcaattggag 180 ctgtcttgtt tgttgcagat aatgtagtca ggactgcctggctgcagaca ctagagtttt 240 gtttaaaaac cgatttcttc ttgtctcttt ctctctcttgcagtataatt acaggctgca 300 gagtgaaaag cattagaact gtttacaaaa cagctcataaagtttaaaat aatggggata 360 cgtgtgtgtg tttgtgtaaa acaaaataat gtgtatggtaggggtaaaca atatccagtc 420 tttcttcttt cactaccccc tgtcaccttc cagaattaagggcatgaagt tgagagatgg 480 agccctttcc tcctgctatg cgatgcttac acttaattagttatgcctac ttatccaatg 540 ccagtttatt gttgcagatc aaaatacaga ttctcaggtgtatggggact gagtggctga 600 tgaaacagac tgctatctaa ttaattttag ggcagcctaaattcccataa agatgttccc 660 tcatgacata tgagaggaag attttatttt tttaatgagccctttgctat ctttccaaga 720 gaaaagcttt cagcaggtta gtgttccaaa gtgagaggggcatttttcca accctttcaa 780 aagcctcctt ctgtgcagct ttgcaaagat tttgcagctcgcccttctgg attttattta 840 tttatttttt aatgcggaag ggtagccgct gactccagcctcggggccaa tcaatcattt 900 tgctttgcag gtttaagatc tgtgacaaag cgaaacccctgtgctatctg tgccttacca 960 gtctcaccaa caataagcct ggtgactgac aatcgagagggggctctgtc cacgtaggtg 1020 ccggcacaag ctgaggacat gagtgggaca gaggaaccagccttgcacgg aggaagcacc 1080 ttttccttct ggtgattgat tgatggggga cagtgaggaggttttcagag actggaaaaa 1140 attgtcccag tcacttacta tgaagtcttt gtcagcagaaaagactctcc ggggtagaga 1200 atgatataat gcagatgaca aatgacaggt gtgtgtttcgttctgcttgc taggtactca 1260 gtatcacacg caggtgagtc agcgctcccc aacatgccccttgcgccatc tgctccccac 1320 atgcaaacac tcgttcccaa cgctctgtgg tttccctggcactgctggct cttcctaatc 1380 gatcgtcagc tctgttgggg atgtgtaaag tactgtcagagtgtgagcag ggtgatacct 1440 taccaccctt ttatggagct gattatgaaa tgaagatagcatttgaatca tttgttagca 1500 gttctgaaag ttgtttcctt ctgttcctcc cttttggagcacagaagaaa aaatatatgt 1560 aatatataca catataatat gctgttgcaa gagactacttcagatcgaaa atctgttttt 1620 aaaatcattg actgatattt cctttgtatt tttttctcccccttccagga ccctatctgc 1680 agatgttctg aatacctctg agaatagaga ttgattattcaaccaggata cctaattcaa 1740 ggtattagct ctcgtcagaa agcttttaca tttgagctctgtgttggaaa ttctattttg 1800 gcaatgaatt gaaataggaa aagttggaat gagaataaaggacaaaagtg aatttgcaaa 1860 ataatcaagt gcttaaaaaa ctacccagca cttgtgagggtttgctattt ctgactcatg 1920 tgcaaccctg tctctgccag cttatgtgcc aatactgacttatttgtagc cctttctctg 1980 caactgtgct tggagtttgg atttcatttt agtctcaccgtctccgtttt tctctgactg 2040 cccagaactc cagaaatcag gagacggaga cattttgtcagttttgcaac attggaccaa 2100 atacaatgaa gtattcttgc tgtgctctgg ttttggctgtcctgggcaca gaattgctgg 2160 gaagcctctg ttcgactgtc agatccccga ggttcagaggacggatacag caggaacgaa 2220 aaaacatccg acccaacatt attcttgtgc ttaccgatgatcaagatgtg gagctgggtg 2280 agacactgga ctcttcactt gttagtctct tttgttcagatgatttctcg agtctcagga 2340 ttatcaggag acattctgag gctttgcact taattattgcacattaacca acaccctagt 2400 ttacgcaatg aacttgtatt gaccataagg catttggtttgtgtttcagc attacttttc 2460 tgatgttatg cttttgaaat ggtcggggaa ggggcctgggggagtaggac aatggagaaa 2520 gagggtcagc actgaagact gtagaaggaa aggattgaaagccctcagtt aagacattgt 2580 aaaaatattt gggcaaagtt gtttcaaaga gtatgaggatgtgactgtaa ttttatgcaa 2640 tggatatgaa tatagactga tactaaagga actttcagtggttattagta ttagagtgga 2700 ttacttattc acagtttgtt atagtaattg ttaggtaattcaaagttgca gtgttctata 2760 tgtcttttgg tagagaatcc acttactact accttagatatgatgctttt ttatttagct 2820 tgcctaggct aagcgtagag cacccagaaa gcctgccaaaatctagtgat tctaacttac 2880 cttctatatc acctgactgg gtttcttacc ttctcaccgtcttcaatggc ccagccctac 2940 agtcttgttc ataagccaag ggccaattct tctagtccacctagtgcaag gcagatagaa 3000 agcttgcccc tagaagttgt cactaccact cctcatttcttttcctgaac ccaaattcct 3060 tgctctcagg catcacccag ctgtgcttag ccatcacattcaacctgact ggtagttgaa 3120 tcttctagca gagcatgctg ggcttcttta ccgagctcctgaggctcagg ttcttgagga 3180 taaaactctt cacgctggca cttggtctcc atggaaggggactttgcttt cccacttgaa 3240 accagacggt gagatcccag taaagttaat tccttgggttcagctggaag caaatgcgct 3300 aaaaagccag cagatgtcat tattgctgac gttggtttgaggagtcaacc caactttttt 3360 ttttttttaa caagggtatt gattttcagg cgacaggccaaaatgaaagg tgtcacacat 3420 acatgagtgt gtatttagca catatgatgt tagtatgtatgtaagtggtg gtttaaatgt 3480 tttcattcac ttacagagca agtaatttta gcttttttagagccttgtgg gtccatttca 3540 agttagttta gtgcctaatg tgttaatagc acagtctctgcatgaggatt gcaatgttaa 3600 acatatcctt gccctctgct tgacctcaca cctgaactcaccttccttaa tatctcaccc 3660 atccatcgct tttgctacag ctgagatctc tggatcctctcatctttccc agtttttccc 3720 tcaccggttt gtcacctggc ctgcctgcct cctctagtcttggcctctct tgcccaccct 3780 tcactcaatt gccagagtta tctttcaagt atctctctgatcagatcact tttctgctta 3840 agtcccttct gtggtttccc tttgccatga gattatgcccttctcctctg tgggcaaatg 3900 agttctgagc cttgcgaccc ctgcctgtcc ccaaggctgtttccctttcc tccactttca 3960 ctctgtgctt cataaacaca attgccatct ccccaaaggtgtctggtggc ttcacccccc 4020 tcgcctctat ccatgccttt gcacatctca tcccctctgccttttcctct ttccccacct 4080 ggagaaaccc tacctgttct tcataaccca gttcatgtcatgcgcttggt gttcccaaaa 4140 caaaaccacc ctcccctcca gcagcattga tggaactttcctttgcaccc cgagaacacg 4200 actccagcat ggtgctcatt ccaccatagc cttccatgcctgtcccttcc acttgaccaa 4260 gatcaaccag agagcaaggg tgtgttttat gttgcactcctcatgcactc ctagtgcctg 4320 ggaatatagt gggcactaaa ctaacttgaa atggacccataaggctctaa acaagctaaa 4380 attcccagaa ataaatataa gtattcatca tcatctacctcttcaatata cagcactgtc 4440 tttaaaatat attaagaaat tgctcataac tttcttttttaacagaattc aaattttcag 4500 ccttgtccta tagttcattt attcatccag taaatattttctgagcagtt accaagtatt 4560 gccttctgtt ggaggcatgt gactatcatg gtgaagggttatagaccagg ctcagccaac 4620 ccagatattt cctacatatt tgcctttaac tccttccttgattttctagc cagataacat 4680 gctaagaact agctcattgg ctgggtgtgg tcattctcgcctataatccc agcacttcgg 4740 gaggctgagg caggcggatc acctgaggtc aggagttcaagaccagcctg gccaacatag 4800 tgaaaccccg tctgtactaa cagtacaaaa attagccaggcatggtggca ggcacctgta 4860 atcccagcta ctctggaggc tgaggcagga gaattgcttgacccgtgagg cagaggttgc 4920 agtgagccga gatcacacca ttgcactcca gcctgggctacagagcgaga ctccatttca 4980 aaacaaaaaa agaactagct tgtttaactc tcacagtaactctaggaagt agttactatt 5040 ctctctttaa tttaggtatg agaaaaatga ggctcagagaagtcatgtag cttgtggtga 5100 gcaagtaaat tgtgaaaccc agacctgtat tagatctgagtctccacagc tacgtattta 5160 accactgtcc cttccattaa tcagccatct acaaataattattaagcacc tcctaaaaac 5220 aatcggcaaa ttcataatat tttaggttct gggagagagggaacagaagc aaaagatagg 5280 ccactggcaa aaacaaaatg actaggaacc acagtgaactgtgggttgga ggtccagaaa 5340 agacctcctt tcttggcaga aaagtcatgc tctcggtagtggttcccgct tgatggagac 5400 ttttcctcat ttttctgtaa tgtgctcata atccttctagaacattattg ctctagattt 5460 tgggtgttgg ttgttttgtt tttgtttttt taccatcttaaccattttta agtgtacaat 5520 tcagtactgt taagtacatt tacattggtg tgtaacccatctccagaact ctttccatct 5580 tacaaaagta aaaccctata cccattaaac agcaactcccgtttctctct ccctcaatcc 5640 tggcaactat cattctactt tctgtctctg tggatttgactactctaggt atcttatatg 5700 ggtgaaatca tacagcattt atctttttat gactggcttattttacttac cctcatgtcc 5760 tcaaggctca tccatgttgt aacatgtgtc agagtgtccttcctttttaa tgctgaataa 5820 cttttcattg catatatgta ccacattttt ttaacccattcatccactgt tggaaatttg 5880 agttgcttcc tccttttccc tattgtggat aatgctgctgtgaacatggg tgtacaaaga 5940 gctcttcgag accttgcttt tagttatttt ggagctatacccagaagtgg aattgctgga 6000 tcatatgata ctcttatttt tattattttg aggaaccaccttactgttct ccatagcaac 6060 tcaccttgtt acgttccctc caactgagca caagtgtcataatttttcca catccatgcc 6120 cacacttgtt attttctggt tttgttttta atagtagccatcctaatggg catgaagtgt 6180 tatctcattg tcactttgat atggttaggt tttgttttgttttttactcc agtgcaattt 6240 tctttgccaa gtgctttaat aaatcttcac ctaatgcaaacctttttgca cctgtaatgc 6300 tgtaggcaat tttggctact gtgcctccaa aaagaaagtaacagacttag taaagactta 6360 gaagaacaat tatgaaaatt ctaatagaat acttttctcactgggaaagc ataaaacaat 6420 cctatctgtt tatctagaaa gatgaaggct gagaagggctataatcaaag tttataaaat 6480 cgagccaggg aggaaagtac tagttcatta tgagcaggagcccttccctt cactttcagg 6540 acaaataaac tattactctg taaagggagt aagacacataaggaattcat taataaaagt 6600 actagaacct tgcatatgga taacacttta tcttttcccaaagaacattt tgttcacatt 6660 attgtgaatg aatattgcca tctcacatga gttgggtgggtaggacaaag atcatcatcc 6720 caggaggaaa cagcctgtgg aaagatgaaa tggcttaaccatggtcccct tacttctgaa 6780 tttgcttttc ccacaactta agaagttgtt cacgttgtgcctatacccgt aagtacttca 6840 gagttttagg ctagaataat gggtggaaga tttacaattttttaagctaa gtaaacacgc 6900 aaggaagtga tcaacataaa gttcaggata atggctaccactggggaaaa agtgatgacc 6960 gagagcatca gtacattcac attctatttc aaaactgaaaaaatatatat ctgaaagaat 7020 ataatcatgt taagttttgt taaagctagg agatgcgttatttattctgt tacaccctac 7080 acttgaaata ttttataaat cagaaggaag aaaataaaaaatgtttgagg gactcctaac 7140 tttctgagcc tagcatcaaa gaaattgggc tctaaatctttcccaaatgg cctttggtgc 7200 tagtctgaga aacaaaatac tgagcagaat agaccattgatctgactcaa catggcattt 7260 cttacattcc taggaaattg acctgacagg aacatctcagtaacattgac ccgatgtttc 7320 tcttagatca tatgtatagg gaagctatga gtacatggagatattttgtt tctttaaaac 7380 aaaagccaga acacaagata ttcagaccag agctagcccacagatgtatg ttgtttgatg 7440 agcatacttt tcagaattaa gccaatattt taaaatcatattttaaaata tatataacat 7500 ataaaaaaat cagattctga cgtttcttga aaattcaaaaaatgaaggct tgtattccca 7560 tgaagcagtc atcacttgga tgtttgaagg ggagctgcagctgctctcgg acactgggga 7620 tcccccacta ctgtgttgtc cctcaataac ttatgccaaggtgcaggtgc catttatcat 7680 tgaccatact gctgtttttc ttatagtcag gaaaagctctataaaccatg tctctatcaa 7740 aggtaaaaca acaaaaagac acaaaaaccc tctgacttttcttatacctg gctgccttca 7800 cttgcctacc ttatctatgt agccctgtag gcatgttaacttccattcct gtaagtagct 7860 tttaatggcc ttgtgctctt ttggaggggc caggaggaaatgatgagtct tagtgatctg 7920 gaaacattcc agactaattt attatcttct cttttttcccttcttctccc aactaccttt 7980 tccccctttg gttttaggta aaaaacatct ttttttttttcttgccaatt ccatagtttg 8040 ggtagaaaaa aaataccatt taattttgtt attttttatatgcttatgct tttagaatta 8100 gaatattcaa tagatcatac aggcatttta ataaaggaaatgtcactgtt tcatgtttca 8160 tactttacta tgaaaataac tgttggcatg tgaaaatggcagggaagtac ctaatacaaa 8220 cgacttaaat atttaaagca ttagccttac ttcatcataaaagaatgaca tcgacaaaca 8280 cccctccaca atcagtaaga gctgggtttg cttgttgttgtcaatcttca aaccatgaat 8340 gctctaatcc acatggaagt atctctctag gaagccaggtgtatatgaat cccacatgtc 8400 tgggatttcc ataagtgata atgacaatat ttactgttgaatttctggtt ctgcgagttt 8460 ttctggacat gacaaacagg tttgatgaaa tttttaatgtttagtaatac aaaatactgt 8520 atactcaaat gctaagatat atgtatcggg aaaggtaaagctttgttttg aagtttaaac 8580 atgtcaatga agtaattcag gatctccgtg tgattttagcaaagtcatcc aaattagggg 8640 gacattttct ccgatccttt ttcatacaaa tgatttttgtatgaaatcat tctgtatgaa 8700 atgattttgg gaatcaagta gttggagtat ataaagtgatttaaattcct cactatggtt 8760 ctgtctagcc acaacctatc acatactcca tccaaagcactattcctgga gctttgattt 8820 ttaactgtgc caaaattgcc aacatggtgg atcttgatggattcctgaag ggtggcactt 8880 ctcccactcc cagccaccgc tgacctcttc agcatcctggtccacagccg atggctgtca 8940 cagcatccgc ctgcaggagc aatggaagat gttctcagtcccagcctgga actggggcac 9000 tgggacagtg tgtcttggca gtcctgtggc ctggaccttccctctcatca agccagccca 9060 gtcagcagtg catgcacacc agctacacag acataatcacggttgcttca gagcatttgt 9120 gtctggctcc cacttcatgg gacaacttaa aataacaatgaagttgggct ttttgcttag 9180 ggtgaggagt cagtgtgtgt agaaaaggga agaattgatatttgtatttg aaaaccaata 9240 tcaacatttt aagtcttaca gcatagaaaa ggtagttgacctctgttctc tcctccagct 9300 tttggacttc agatgtagat tacagagaag aagatggaagaggcaggaga aaatttaatg 9360 tgatggggtt gaggtgagca catagggcaa ccacagccacctcggcacac tccttacagg 9420 aagtacttga tggcatcctg ccgtttgctc agcaacaaccttgtaaacta gtcggagtgg 9480 ggagcgctat tagaagctcc attttacaga tgagaaacacaaaccaaaaa aggataaatg 9540 acttgctcag gctaagctgt ggcaaaatcc aaatgttttcccacatgttc aagtctaggg 9600 ctctttccct tctcatcttt atcatcctca cccctgccttgtgaatctga ttatgagtgt 9660 tcagctttac aattcatatc attcctatga aataattgttgtaataactg agaatcagca 9720 ccgtttgcat ttcaggcacc caaggcctga aacctacagaagtgaagagc ttcagcttaa 9780 aacaaaactg agcccttcca cttatcggac agacattgtcctgtccactg ctctcaacag 9840 ctgtgaataa ataactatga gttttattac agaatcatttttagtaaggt ttgccaataa 9900 aaaaatcaat ggcacaatca cagtgggaat atatacagggtaaggcagtg ttattgactg 9960 acattaatca ttgcacagcc attaaaaggt gctcgatgctttccttactg gtacacacta 10020 acacagtgga gtgaagagat tcagattcct gcacaccgtcatctgtctct acccagagaa 10080 tccatagaga tcagaggcaa caattaacgc caccatggcagtcagcagct aaagacagac 10140 agatccacag cccactcatg cctcatctgt gcctgagccactgagaccat ttcagccttg 10200 aggggagggt aggagacagg tgttaatgtc agtcagtatctcctctaaga ggcgagaaga 10260 atcaaaagat gagagtgcag aaagagagac ctatcgcgactgcaagcaga cgtcaattct 10320 gtttctcatc ttgaaaatgt caatgtcaca gccagcattcagcctagata gagctcaaga 10380 gcatggtctc tgggaagact gcctgcattt acttctgggttctgctactg agggtctgtg 10440 tggccctagg cacagcatga tttagccttc tgcacctccatttcattatc tgtgatatag 10500 gaataatagc tgctagcacc caccacaaag gattattgtctggatcaaat taattgctca 10560 tgtgaagcac ttagaagaga acctggctga gagtaagtagaaaatcgatg tttctttttc 10620 ctgtctacaa tataccttat agtcttggca gtgatctaagccacctagat cactcaaaat 10680 taggcaccca ctacagtctc ttccagctct aggcttccctgattctgtgt ccaacctaga 10740 atactttttc aggtgagatt cactgagagg cagcttcggcttcacccagg tttcattaga 10800 aacacaaatt ctcaggcccc atcctggact tactaagccagtatcttggg gtgggtacag 10860 gattctaggg cttattatgc ccttcaggtg attttttaaagttggacaag cattgtctca 10920 gaatctcttt actgagaacc ttcaagccag attcccactttttgttttgt ttttatgaga 10980 cagagtctcg ctttgtcccc caggctgaag tgcagtggcgcgatcttggc tcactgcaac 11040 ctctgcctcc tgggttcaag caattctcct gcctcagcctcccaagtagc tgggtctaca 11100 ggcgcatgcc atcatgctgg ctaagttttg tattttcagtagggacaggg ttttaccatg 11160 tcggccagac tggtcttgaa ttccccgcct caggtgatttgcccaccttg gcctcccaaa 11220 gtgccgggat tacaggcatg agccactgag cccggcccgagattcgcatt ttcaaggacc 11280 ccagtggtgg agctgtattc ttaacagtgc ctatggggatccactgttgt cttggttcct 11340 tttgacgtgt ggactggtgg gagccagggc atctggagggattgcttggg agccctacac 11400 aatcagctca gttagttaag aaaaggatga tactgagaatgagaggagaa tcctagctgg 11460 ggcacttcca gcagtgatga attgggataa gttttagcccctgtttttat attattttga 11520 ctttaaatca gatttctacc tcggtcccat atttgtagtctaaatgagtc tgtaaactag 11580 agattacagt gctgcgattc agaaccttgg atgatggcctcagctgcatt tttcttttat 11640 ttgaattctg ctaaggctca taaaggaaac tgggagcttgttctttgaaa tagactactg 11700 gacactgaaa agccatcaac agggtttgct gttttccccaaaatcaagat actcagtaac 11760 taactactgc agagatctaa agaggagcaa tgagacatggtagaaatctt cagtgtcacc 11820 tttcaccctc tttccacttc tacatctccg ttcatccacttatctccagg atattccccc 11880 tgtgtgatca accaacacaa atataaaatg caacatttctaaaatggggt tttaaaatct 11940 cccataaggc atattccttt ttagttactt cttcaccatcagtgaaacga ttggtttcca 12000 gttttcttct cacggtgaaa acttcaaggt gttagccaactcccagcctt ctcatgcatc 12060 accacatcac ttgggtctgc ttcttagggt cgctctgtgtcagaggcata gtcgtccgag 12120 ttcttctttc ctcaggggta gacggcttca gcgacctcatctgcaccctt cgtggaccag 12180 cagcagtgtg ccctcctcaa tccacacacc accattgcaactgcaacgcc tctcgagggg 12240 attgcaatcc actgcagcag caacacctta caaagacttttttcttaaca tgccgttcgt 12300 gtctttgaca cagaacataa ctctaccttt gttttttttattctatcaca tccagtatct 12360 ctgcccagcc ttcaagctat ttgctaaatc tgctttccacatttcatgaa gagaattctc 12420 catatttctc ttcatgccag gaaatcaatg tgttgttctcatcactctac ttgtttcttc 12480 ccacctccag agcatcacac atgtcactcg ctgcaatcccccaatgatat acttcaccca 12540 tccgacattt acttcctcct ttttttccat ctgtcctcatctctcatttc agaactctta 12600 ttaagccatc aatccaagtt ccctctattt ttatatgccctgctactgct gtattcacat 12660 tgttacgtgt gcattgcttt gcaaatatcc ccaagtgatttttctatgta ggttctcctc 12720 tagagaattt attactgaag accagaggca gaaaatttcccatttctttt gtttatgcta 12780 cacttgggag atcagcccgg aatgctgagg ttgatagaatgttttgcatg aagtaggcac 12840 tcaagacatg ttattcattg actacaaaag gttgcttgatgccctgaaga caaccagaag 12900 agaaagagca gttaggattg ctcctagccg aggcatattttagggagctc taatgagacc 12960 caattagaaa ccctcctcct gttctcatat gcgtcatgtggaacggtaaa agctagtata 13020 tgaagacacg gggctaactg tggctttttg gactgttgcatgcttctctt ctgccctgaa 13080 tgcttgcctg gtttgcatta cgatttcctt ccttgctggaaaacatagac tttcttcctt 13140 tctttggaag aagtgggacc tgttggaaat ctgaggctccaagattcaaa tgcaatgggc 13200 ttggctgtag tgctctgtag tacacttctt gacaagtcctgaaaagacta ttgaattctg 13260 gcctgtgctt ttctgcagtg tttggatgtg tttcagctgcagtgtttggc tggacagagt 13320 aatcgaaata gttttttttt ttttttcctt cctcagcaatcttacgttac cagttgatcc 13380 ttaaagttaa aatggaatat tttacaaccc tgcaaatactttcgagtgca atcgaattat 13440 agctctttca caagcaaaca gcctatcttt aaaaaattgtgcataaatag gtcaaaatat 13500 aaattgatgt tgttatccta atagaaaaac tggcaaacatttggtgagct tgctagagga 13560 taccaacttg cattgaagat cttttttaat tattattaaacaaacacagg cattttgatg 13620 ggacttaata tggaagaaat ttttttctct tttctttcatacgattaaaa tgctatagta 13680 gtaatctaca ctagtaatct agtagcaatg tcactagtagattgcatttg tgctgggctg 13740 ggggttttga tgctctgtgt atcaggctat tgttctctggtaatcttcaa agggctgctg 13800 gcctttgaat ctgctccata tctaaaattc tagctttaattatcagatta gcctgcattt 13860 ttttttcttc gtgattacag ggaggagaag tgtatgtttattattctgca aaatacttga 13920 gatggtagga tctcaaatgc ttaattttgt tgtagaaatgagagctgatc aaattataga 13980 ctcctttaag ctaaggaaag gaaatgaata agtcagggaaacagaggcac tgaggggcac 14040 aatcgaaata ggcattcatg tgctgcactt tgctaaacaatgctggcact gtgcctttca 14100 gggtccctgc aagtcatgaa caaaacgaga aagattatggaacatggggg ggccaccttc 14160 atcaatgcct ttgtgactac acccatgtgc tgcccgtcacggtcctccat gctcaccggg 14220 aagtatgtgc acaatcacaa tgtctacacc aacaacgagaactgctcttc cccctcgtgg 14280 caggccatgc atgagcctcg gacttttgct gtatatcttaacaacactgg ctacagaaca 14340 ggtaagggat gacgtttcta gcccatgaac gtcttgtaatatgtcttaga ctcaggaaga 14400 agtgtcatgt aatgaaatgc atgaagttcc aacaaatacctaaaaaagga tcaagtgttt 14460 ttaaactgct tgacatctac cccagggtct gggacacagtaaatgatcaa aattatttat 14520 tatttattaa ttaatgaaaa actgatgggt aaatcaatcatgtgtacctt gttgattcat 14580 gtatttgttc attcataatt aataggattt ggaaagtttctttagcttgt ggttgttttt 14640 aggtctcaaa tgttcacttc taccttccag gaaaggaagtcatcctaatt gctacacctc 14700 ctattattgt ttaacctaca cagggcaaga agaggtttttgattaattgg ttttttgaaa 14760 attggggact tctttcaaga ggggtagtga acatcatgccagtctttctg agaaaaaagc 14820 aaggttcctt ctggtaatat tagccccaag gccctatcctcccagcatgt agatgatgtc 14880 cttgggtttt ttgtagcatt tcttcataaa agggcacaacgttgttcgta gaagggctac 14940 agtgcagaca tgggttgttg cttgttttta tttttccagaatcacctagg cttattcata 15000 agaatcaaat atgttatgat taaagcttgt tcaaatgactttcaggccta caatttctct 15060 attttgaaga tttaaaagta aaggagttca gataaatattctcctgcatc tcttttaaaa 15120 gtgaaattaa tctttcccac tggactcagg aaatatatggccatttgctt ctttgcagag 15180 cgccctacgg gcacttaata cttgttattt atggatgaaaatattgattg tgcatatgat 15240 agcactgtca ctcgcagaca gctcaaagtc tcgactcgaagcagctcctc ccgtgcatct 15300 caagggtgtt ttcttatgcg taggaaagaa actaggttacgtagaggtta cagagagtgc 15360 cttatctgag tgtgttttct cacattagtg atttaaatttatagcacctt tcatttgagg 15420 actctaaagc aatttgcaaa tccaccaaac atccctgtagagttcataga tggcaaatgt 15480 acttggctct gttttataac tgaggtgggg atttcaaatattgcgtgacc agctcagaaa 15540 cagaatcaga ggcggttccc taataaccgg ttcctgattttaaccatcaa attgtgtttt 15600 gcttctggga agtgttgtgt gaaatcagtg cctttcacctcccaggttcc ctagtagcaa 15660 acatggaact ggaacgattg ggactctcac atatccagagtgaaatggat gacatttgtc 15720 ataagaaata gagattgagg gtgagacagg gccctgccaggacttagagc agtcaactag 15780 ggtttgctaa tttgtttcag ggctaaatta ggaagggcaagaaagaaggg aactccattt 15840 tgccattatc tcacctaatt cctagggcaa tcttgaaagaaaggaatttc ccatgttact 15900 aatgaaaaaa ccgagactca gaaaggctaa ttgtctatggtcatgagcta gtcggtgatg 15960 gaatgaaccc agcacatctg actccaaagt ctttcctctgttcattgtct tatttattta 16020 tgtaatgatt ctcttctgcc tcattccaaa acgccttccagagatacaga ctttagaagc 16080 tacagaccca tgaggaccct ggcaaacaga aacaaagagtaggactagct aaagtcaagg 16140 caaagattgc tgtacaaact gcttgctgtg gagttgtagggaaaggtgga atatttagaa 16200 ctaagcatcc agatgttaag agcaaaaatg gaaacaccatccattgtaaa gttcacccag 16260 tcaccgggat aaaaacaatt cagttgcctc taagcatgtttcctggtact tgcttcggtt 16320 atactgtgct atatccacct taagagggat tgagagagtttaactaaaac tcaaagatga 16380 tttagttcca tttccttatt tttagaggag caaatttacacccacaagaa taaagtgact 16440 gaagtgacag atctgactgt tttttattta tttgtttatttattttattt tattattatt 16500 atactttaag ttttagggta catgtgcaca atgtgcaggttagttacata tgtatacatg 16560 tgccgtgctg gtgtgctgca cccattaact cgtcatttagcataaggtat atctcctaat 16620 gctatccctc ccccctcccc ccaccccaca actgactgacgttccaatgg gaagagccgg 16680 caccgggacc ttagtcttca gggctctctc cagtgtgctgatcaaacctt aagaatcggg 16740 tctgggatga aactgtgtac acaccggaag gcttccctgttacctcaatg gactgtcact 16800 tttttgtgca gcccgagaag tttgtttcag tcgatgtcttctccagggag agtagatttg 16860 ctcctggaaa ctaaatctgt ttctaaacct tttccccagggtttagacac ttaggtaata 16920 atacaattta tagtgagtca acctccaaaa taaaaggaattctttaaggg aaataaagac 16980 ttttccaaaa acttagttaa cagtaggaaa aggggctgggtgcggtggct cacgcctgta 17040 atcccagcac tttaggagga caaggctggc ggatcacgaggtcaggagat cgagaccatc 17100 ctggctatca cggtgaaact gtctctacta aaaatagaaaaaattagcca ggcgtggtgg 17160 cggacgcctg tagtcccagc tacttgggag gctgaggcaggagaatggcg tgtacctggg 17220 aggcggagct tccagtgagc caagatcgtg ccactgcactccagtctggg agacaaagcg 17280 agactctgtc tcaaaaaaaa aaaaaaaaaa aaggaaagaaaaaccagtag gaaaaggata 17340 atatgggctc aggaaaacaa atctagattt gcctattagtaaatcagaaa aaataaaaat 17400 tggggccggg tgcagtggct catacctgta atcccagcactttgggaggc tgagatgggc 17460 gtatcacctg aggtcaggag ttcgagcctg gccaaaatagcaaaacccca tctctactga 17520 aaatacagaa attagccagg tgtgttggtg catgcttgtaatcccagcta ctcaggaggc 17580 tgaggcagga gaatcacttg aaccaaggag gcagaggttgcagtgagcca agatcgcacc 17640 attgcactcc agcctgggca acaaagcaag actctgtcacacacacacac acacacaaaa 17700 aaaaaaaaaa atgggaagac tgaaatggca gtgatagagatctctataat ttaatttatc 17760 cttgatggga aaacattaca gtatctcaga caattaggaagtaaaaatgt cttcatgaca 17820 atagttattg atccaaccat ggacaacaac ccatttgaaaatacacaaat aagtacacac 17880 aagtggcagg aatcttagaa aattaagaga attggtaggtagatattaaa ccaatgtaat 17940 tatgagacac taaactagta gggagaaagt caatgtgtaaagattctatt gctttcctaa 18000 taatgaattt attatttgga acatttagct acaaacctaagaaaaataat gacggaaact 18060 gaaatatgta ctatacatac ataaatatcc atctagagagtaagttttat gtagaataac 18120 tcagagaaaa caaattacta aagggcttat tttgggaggcgtccagaatc catggattta 18180 aatgagttga attaatccaa tattatattt accattgtatgacatgagtt ttctctttca 18240 gaactgaggc atatttttgg actggctata gcctaaatttttctaagttt actcatggaa 18300 aatatgagtc tatgagagct tgaatgttca agaaggggaaaatgcagtaa catgtcgact 18360 gcacttcata ttctgacaag tgaaatagga atgagattggattatatatc ataaaaatga 18420 ttatcctgcc tgaaatcagc tccaaaaaaa ttaacttaaatttattatga ggatatcaac 18480 attaatataa aacctggctt aggttttaag gtaaaaataattatccatgg ttgagtgata 18540 gcattaagta tttaacaaca acaaaaaaaa ctgaagcaagattgaaaaca tcgtggaatc 18600 acattctacc ttgatttgtt ttgaggactt ggactggccagttttccaat ctgtatgatg 18660 cagagattgg cctagaatga ttcctaagtt ttctttcttgtccaaagtta cagtgtgtta 18720 taactttatt atactcatgg taattataat attgattcatttaacaattg tggtatttga 18780 attataatcc taacacatgg tacataaaat cacatatgagttaaatctta atcacatgaa 18840 ttctacctca catcactgag gtagaaggga caatatttaatagacagctg caataaatat 18900 ttttgtgaag aattcttttg tcataaatca aaatcatagaccttagttct gaaaaagaaa 18960 aattatatca aataatggta aatacaatat tggatcaaaaatttttttgt ctttttacgt 19020 tacaaaattt atgttactta tagcaagtat atttattctctaatatgaga ttttttaaat 19080 gtagagttca cttaaagtaa gacaaactaa tattcttaattttattatga tgtagattct 19140 tgatacatgc ataaacatga gaaatgtcat acatttattaaaccacagtg tgcttggaac 19200 acactagaca ttgggatgaa gacatttaaa ggaagattcctgtcttcact agacttacaa 19260 tctagttgag gaaaccagac tgctgtatac aaactgacattaatcataat tttaacttgg 19320 ttcaaaatta tttatattta tagttactag cgtgaaatccatcaccctaa gtctatcaat 19380 tacgtggatt aaaatctcaa tatatctttt gatacattaaataagatttg acttttctgc 19440 ggcatcagat ctttgggtta gtcactattg ctggctttaaaagaaattcc ttggcttcag 19500 gtagttcctg gaaatttttc taagcattat ggaacaggttgtcctagaca gaagtagcat 19560 ggcctgaagc caacaataat tacaatcagg tcttctgatctttctccctg ccccccaacc 19620 cccaccacct tcttaaacag ctgtgaaggg aagtgcttaatggtatccaa aacaaagagg 19680 atgggtaaat ggcacattag tgatgtattc agatagtaggagttgaattg aattgccaat 19740 gccgaaggat agaaaaatat tgaactatac gtaacctacatgtagacata atggcagtaa 19800 gggcaagaaa gctaaattca ccttaggaag ggaaaaagagatttaataca tctggaggaa 19860 aataattaga aggccagata atcaattgca gagcgccgccaggaaacatc gtgttgaaag 19920 aggccggggt gattacaaac gagtctcaat gtcatgaggcaacaaaaagg ccagagcaac 19980 tggaggccaa cagtgctgca ccctgacacc caaggcccccatcagccttg gaatgagtgt 20040 gatgggtgag cgcacatctg gaatactgag tacatttcttacgctccgtt aacacagaga 20100 caccaaaaac ctggagggag ttctgaagca aataacaaggactattaaaa gacttgaagg 20160 aatagtttat aagggaagga ttaagtcaaa ggggaatgccatggcaatgg gtagaaaaca 20220 gtacaaaata tcctacaaag taaaacaatg aggaaagggcaggaatgact tggggagagg 20280 aaacaaaaaa accccaacaa tgaagtaact taaaagtgcagaaaaaaaaa ttaaaactaa 20340 ttaagcagaa aaatgtaagc caaatggagg agtttgttgccacaaaataa gtagtagtgg 20400 ggaagaaaat atgtaacctc cgaagagata ttttcaagtgcacaagtgca gaactctagt 20460 gcgagatttc cttacactgc aggatggaaa atcatttacaaaagacaggg ccaaaagaat 20520 actgctaatg gtgatgctaa taacaatatt agttgtaggagcacttaaca agccgttgtt 20580 ttgtgccagg cactgttttc agcgctttac atatgtgttgatgcatttaa tcctcaaaac 20640 aatcctgcca ccattattat tatcaccata gtggctttgcagaaggggag ttgggggagg 20700 gagaagtgaa gtaacttgca tgtagatgga taccctagcaagtagcagag ccagaatttg 20760 aacccaagca ggctggctct agggtttata ttctcaatcactatgctttt tgccttcttg 20820 gaaaaaaaaa aaaaaaagga aagaaaagtg ggataaacccgtagggatga ggaggaggcc 20880 aaggaaagca cggggcttga ggctgttaag tgcaagctttttggaaacaa tcgcttttga 20940 acgttagtgg ggtgtggcct tggtctgctg cctgtggctccaagtcatac tgcattttgt 21000 tggaaaagga aaatcatctt gtggttctat gtgaaaaggtcagttcgtct ctaagacagg 21060 aattcctcat taaaagaatt ccaactacac gtagtcagcacagaaggaaa tcctgagtca 21120 cctgatgtga gaccctttga cactttgccc tacactgatcaacgtgctca gtgcccctgg 21180 cagaatgctt aagcagcggg cacttggctg actgtagacctaattggttc actcattcac 21240 agagccaaca aataaacatt cattcaacaa acaaacattgccatgtttct cagactggag 21300 tctagattct tttaaaaata atataataag aaataacaattttagaaact ctaaagctct 21360 attctatgaa aatgttttga aggccaaatc agctttaaaaaatatgatga tttgattggg 21420 cgcagcgcct catgcctgta atcccagcac tttgggaggcctaggcagat ggatcgatca 21480 cctgaggtca ggagttcgag accaacctga ccaatatggtgaaaccccat ctctatttaa 21540 aaaaaaaaaa aaaaattagc caggtgtggt ggcgtgtgcctgtagtccca gctacttggg 21600 agtctgagac aggagaatcg cttgaaccca agaggcagaggttgcagtga gccgagatca 21660 caccactgta ctctagcctg ggcaacaggg taagactccatctcaaaaaa ataaaaaaga 21720 taattcaagc aaaatcacaa aatttttaaa gtctagacctcgtaaagtcc ccagaataca 21780 ttggattcat gaacccaaat tcaagaaaca aaaaggatggagccctgaac tgtgtgcaag 21840 gatggagagt gcctcagaga taaggcagag gcaatgtttgccctcaaaaa gcttacagtc 21900 tagcaggtgt tcagcttcta tatgaacatg actataccacaatggagaaa gggaagatga 21960 cattacaacc acaaagacag tgttgtggaa ttaagacagagactgtgagt gaaatggcat 22020 ctgctctggc cttgatatat aaagaggcaa ataaagagaattgcacaagc aaaaatagag 22080 aggtgggaac cagagagcaa atagaggaaa cattagctggagagagggac gattaacaga 22140 gaaataggag atggggttgg aaagggaagg attttgtccaaactcaaagt aggcctctga 22200 gggcaagcta gcagagtaca cttgattctg caggcaatgagggtaatctg agattgcgag 22260 aagagggtga agtaaccaga gcaggtcctt gggaagattaaccagtggca atcgaagtgg 22320 aaagaccctc catcctggct gggaaagtca gtgagaccatgagcatctgt gaggggtggc 22380 aagttaccag ggatggcagg agggatttga gcactatttccaaggcagac ctgataggag 22440 gtggcaactg cccagcaagg ggagcgcagg agcaggcgaaagcagagggg gctctggagg 22500 gccaagcatg gttcatggag ggtgattatg ccattcagaggaatggagta aggctgaaag 22560 ggaaaactgg taattccatt ttaggcaatg gcattaggaagcaagtaaaa cattcagatg 22620 gaggaattct acaggtacag gtgctccttg acttacgatggggtgacatc ccaataaacc 22680 catcataagt tgaaaataag gtaagtcaaa aatgcatttaatatactgaa cctaccgaac 22740 accagagctt agcttagccc agtctaactt aaatgtgctcagaacactta tattagccta 22800 cagttgggca aaatcatcga acacaaagcc aataggcttgtaataaagta ctgaataact 22860 cataggattg attgagtact gcactgaatg cataccaattttacaccact gtaaatgtga 22920 aaatcttcaa ttgaaccatc gtaggtcagg gaccatctgtagtcgtatat caaagataaa 22980 agcaagctaa atacctatcc catagagaca tcaaaattatgtacatcatt aagtttaaaa 23040 ttcagaatgt gtgttttaag accaatgtca ataaagtgctgcaattctag aattcgtttc 23100 tattatccca agccagtctt ccaggaacta cttttttaccatggatataa gcgagggcac 23160 ctataaaatc tgtttaatga agccaggcat tggctttgacatggaaggcg tctggcaaca 23220 gctttataac atcagaaaaa ctaaaacttg cctacatatgtatatgcatg cataggggtg 23280 tgtgtgtgtg tgtgtacaca cacatatata catacacgcacacatgctta tacctataca 23340 gacatatata aaataaagtt ttctctagcc ctttctacttgaagggaaag ctatgtgtgt 23400 ggctggagtg actaaacatt taggtttacc cagaatcatgcttgtttata cctgcttttc 23460 tgtaattaca gcaattacaa ataacatcct cttgctctctcaaaagtatt ccagtatatt 23520 tatgactacc attctgctag gttgtaatgt ctttttcacttcaagaatga acccatattg 23580 ttcctggaat cccagcttct tctttgcttc ccgtacccctctcctgtcat catcttttgc 23640 agaagaccaa atttctagtc accctctcag agagaccgagtcagccctgt ggcacagtgg 23700 tctttcttgg aagtgacatg ccaaagttat aaatgtgaaggccttccagt ggctttttta 23760 gtgaactgtg gtgtctttgt gacacataca cttctactatactataattg tatgaaaatt 23820 agtaatctat gtagtaactc tatgttgaca gaatttttattatcgataat agatgtatac 23880 attcataaaa tacacataac ataaacaccc attacatactatacatgtga tataaaccct 23940 gaccatatcc cataaaaatg gagtttacca tggttccctggtttggaaaa tttgtactct 24000 ctggatatgt aaaaacgaaa ataagctttt caatagtgtttttataattc acaattctca 24060 aatagtaagt tagaaaactt atcacaaaga ctgaactttcagttctccaa cacctgcccg 24120 gtggttgcat tccaaatctc acgctacttc tctgattgttccatcaactt aacaaaagag 24180 catagcctga ttttactcca gtaggaccat aagaaatgaatgcacccaga gtgctgtgat 24240 cattatgatg gtttcattga gctgtaatcc atgtacttggatactacttc tatttatttt 24300 ttaaaaatgt gtttgtgtca ctttgccaaa ggattggagtattacactaa tgtcattttg 24360 gcattcacta ttacctaggg caacttttgt tttaccgtctctttttcaag tcataatttt 24420 atacttatcc atttatttat gattaatcat tttacgtgaaaaaaataatt cttttttccc 24480 actgcagcct tttttggaaa atacctcaat gaatataatggcagctacat cccccctggg 24540 tggcgagaat ggcttggatt aatcaagaat tctcgcttctataattacac tgtttgtcgc 24600 aatggcatca aagaaaagca tggatttgat tatgcaaaggtaattttcag gcacttttac 24660 actgcatcaa tttactttgt gcataatggg gaaaagccattttcagtgag ttaaactatc 24720 cacaagattg gctttctatg ttctcacaat gttagcatgagaaatgttaa ggtaatttta 24780 aactctaggc aaggaaaaga ctctcaagga acgctgcctttgtgtagtga tttccctcat 24840 taggatgaaa ggcaatcagg ctttgatgaa agtatcatcaagaaaatcag aattctctgc 24900 tctcttatga taatttttgt cctcccagtt cccccggacccaaccaagga cttgtccaca 24960 taatcaaatg ttcatcttgt actgttttac ttttcactgggacaaaagta tattttgtct 25020 gtggcttcag atttaggcac aagcataaga gcaaataaatatgataatta aagtttgaaa 25080 aaccacattc cttgctttta ctcctgtctg accaagcttagtatacgtga caaggacacc 25140 ttccctatca cggcaagcat ccacaaaagt ctctaatgctatcaattcta ggattttcaa 25200 atcagttcag agaaactgaa atcaacatgt cccatagttctttgaccagt gggttctagt 25260 tttgacttaa aaattcacaa agattttgtg atagctgacttaagtttaaa tttttttcaa 25320 atcataagaa tgaaggggaa aatatcttcg aatttagcatgcttatttgc caaaatatcc 25380 ccttcccttc cagccatacc catctcttct tcatttatctagaagaagcc gagaatctgc 25440 tctatctagc aacctctccc aacaggctag atcacttggtagaaatcgga aggagagaac 25500 ttgatttaat gttggcatat tgctgtcttt atgcttggcctgatttgagc acaagggact 25560 tgatgggaga taagattaag tccagctcct ttatacccttcagaaaacaa tgaatgcaaa 25620 tgaattcata aacattgcta ataggcttcc aaactcatgaaagttaaaag ttagcagaga 25680 ccttggaggc aaatttgagc aatgtcctca tttgcaaagatgagaaaaaa gcattctaga 25740 gtggctcaac cactcatcct aggtcatatc cccagctgtggatacaatca ttgcaagcaa 25800 atggtgcaga ccacgtgcac taattgtcac tgctctcctttgctgtctgt agggatgctt 25860 tttcatgctc cttgttcaag ttattaacct ttctttccctgctgtcctaa agagagcaaa 25920 gtaatcaaga ttctctccaa atactaaatc agcgtaacttgttcattatc acagctgatt 25980 aagtgtcaaa gacaactgtg tctgaaaaga atatatatctttttttagtg aggaaaagaa 26040 tgaaacagac actcccttgg aagaggaagg ggatagcctgtagacttgcc ctaacaatga 26100 catgcggcac acaccatccc tctgatactg cttttgcagctgttctggtc cttaaatcca 26160 caacatgtga ttagccatgc ctggaagcct tcaacatttgcaaatattgc ctaaacactt 26220 tctgaataaa gtttatactg gagctccaag ccaatgacacacacttaaaa gaagcaggtg 26280 gtttaagttt tcatcttttc tttccttttc attccatttcctccctccct cttacagacc 26340 tgcatcagcc ccccctgact gtgggttaag tcattttattagcaagtcag gctctaatcc 26400 cagcagctgt attgctttag ttgtgcaatt aacacagtataatctgcagg aaatcaactg 26460 ctccctattc aagtgtttca agtaaattaa ctgatcaaatgttgcagctt ttccctgtgc 26520 tcctggattt tggccatggc tttgattact gattattgtaattcccacag gtggattttt 26580 cgtttgaaga aaatatcttt tcttgtgttt atgtattcatgggcgtgtgt gtgtgagcgt 26640 gtgtgtatgt atgtgtgtgt gttctgcaac tgtaaatttgaagtgggcgt gggtgtttcc 26700 tgcccttaaa gtaattaaat tttttgccaa ggaattacatcaatgaaacc tgagactgaa 26760 atatgtatcc ggtgtttcat gtgttctagt acttttatcgccagattaat cattatcttg 26820 ggcaaacacg acttgacttt ttttttcccc attgctaagttgtgtattac ttaaaatcca 26880 tttttcgtat gttaccaagc tagcaaccct agaaaacaactggcagctga ttttctctat 26940 tatcgaaaat gttcggctgc cttgggaggt gcagccttccttcctgctgt agaccttgcc 27000 acttcgtgca gtgaattgct tctgaggaaa gcagttattcaaatgcgatc tgatgaatgt 27060 caccttttgt aatttttgtt ttgtgtcaaa tgtatgtttcaggactactt cacagactta 27120 atcactaacg agagcattaa ttacttcaaa atgtctaagagaatgtatcc ccataggccc 27180 gttatgatgg tgatcagcca cgctgcgccc cacggccccgaggactcagc cccacagttt 27240 tctaaactgt accccaatgc ttcccaacac atgtaagtaacaaactcaac tctgcgacct 27300 gccgaacatg cctttccctt ttctcctcat cccactcctctcctttaccc cgtttccttc 27360 caccctgcgt atccacaagg ctttcttcat gaaaggataacttaagagca gaccacggaa 27420 caggcagagc cgctgagcct gaaagaaagc gccttatctgggtggtttga ggaggaatca 27480 aatttccagc atttacaagt agctaaatag aaaggaagagatgcacatag agtgaatggg 27540 ggcaagtttt acaagagttt cctttcgttg tcttaaataatattcgtgtg tctgatctaa 27600 taatgatgat gatcaaatag tatgcttttc atagctgcacagtggggacc tctggtctgg 27660 ttatagaaac atggatttat tttccaggcg aataccgtagcagctttgct gcagacgtgc 27720 aattagaatt cctgcagaag gcagcttgag tggcttgcccaagagggctt ctcaggtcac 27780 agctttaaaa taacctgatt tttttttttt taaagaggcaggagtcttgg agatgggggg 27840 tgggaaggca caagggagag ggctgatggc gtggagggatgagacagaac aaagagctgt 27900 cgtgtgccca caattctcac cagccaaagg tggaaaaatctagatgcttt ggcagcaaag 27960 aacatgattt tgttgttcac tcagttgaca ccatttcttcctaagctttg ccatcaatat 28020 ccagtcttcc acacagagca gtggagttgg ctctgtgtctgctgaaagcc tgaccattag 28080 ggagacaggg aacagaaaat tggtatctgt ttcctatattgtgaaacctc caaaattggt 28140 tcttaatcta tttgtactta aatatcatct cttttcatccacactggtta ttagccaaga 28200 ttccaggcag aaagaacctt acgaaaatag gtaagtaactatgcaggctc tctagttgcc 28260 ggtcactata catccctaga gaagttttta taaaatgttctctttttttt gagacagagt 28320 cttgctctgt aacccaggct ggagtgcagt ggtgcaatcttggctcactg caacctccgc 28380 ctcctgggtt caaacaattc tcccacctca ggcttctgaggagctgggac tacaggcaca 28440 cgccaccaca cctggctaat tttttgtatt tttagtagagacgcagttcc accatgttgg 28500 tcaggctggt ctcaaactcc cctgacctca agtgatccacccacctcggc ctcccaaagt 28560 gctgggatta caggcatgag ccaccgcacc cagccttataaaatattttt atttgtacct 28620 taatgtaact gattgactta tgactcctgg tcagtggtacacagatcatc tctatgatat 28680 catgtgactt agaccagaaa gaaggaggcc agagctgactcaggacaaga actaacaata 28740 tgaagccagg gtgggttacc tactgagcat gcccaggaactcagaggatg gaagtgtttt 28800 aatgcataaa atatcatcga caaatcatga aggttgccccagcacctggg aatatagctg 28860 ggataagcca ttatgttttg gagtcaactc catgggtggatatttaagct tctgaagatc 28920 ttcccctata tacaactctg cgagtaaatt catgaatgaagcccatgtgt gacaagtggc 28980 tctccattat agctcactta caaatttagt agccaactgattcaatgaaa ggaaaaagtc 29040 ctgcgggctt tttcaatacc cctgaacccc cctgttcccatttctgttga atcagaaatc 29100 actttaccta tctttgttgc attagcagaa acccagtctaaggtgacttc ctataactgt 29160 aaactttaca gatgttccct caagctggag gagaaggggttgacaaaaca gagtgttttg 29220 tggctcctta aaagtcagcc tgcctttgaa gctttgaggcaaggtcctaa gcctgcagga 29280 aaatcagcct caggtcaaga gtttataaga gctcagttgcatggaatcag tactgcatga 29340 ggggaggagc ctgcagagtt ctcagggtct cagcaatagctttttgaaaa acatctctgt 29400 gctggccagg cgcggtggct cacgcctgta atctcagcactttgggaggc cgaggcgggc 29460 ggatcacgag gtcaagagat caagaccatc ttggctaacactgtgaaaca ctgtctctac 29520 taaaaataca aaaaaaaaat aaaaattagc caggcgtggtggtgggcacc tgtagtccta 29580 gctactcggg aggctgaggc aggagaatgg catgaacccgggaggcggag cttgcagtga 29640 gccgagatcg caccactgca ctccagcctc ggcgacagagccaagactct gtcttaaaaa 29700 aaaaaacaaa gaaaaaaaaa gaaaaacatc tctgattccagtaattaaaa attctatttc 29760 attccacgaa tatttatcag tgccacatgt gacactatgcagcccagcag ggatatagat 29820 aagcgtgagg aagacacagt tgctaacatt taaggacagataaactaagg caggggttgg 29880 cacactggta cccacagtcc aaacctagct tgccaccagtttttgttaac aaaattttat 29940 tgacacacag ccatgctcat tcatttatgt attgtctgtggctgctttca caatacaaca 30000 gccaagtcga gtagttgtgt cagataccat acagcctggaaatactatct catcctcgat 30060 aaagtaactt tgccccaacc tctgctctag gggcaggatgggcaagtgtc ccaatggcaa 30120 tatcaccaat agggccagaa gtgacaagca cagagcagacgttcgcaggg ctgtggagcg 30180 gggagggaga agccttcata tctttaaagg aaaccaggaaaacttcatgg accaaggctt 30240 caaagtgggc ctcaaaagat gggtaaaatt tctgcagataattgtttaga gattgggttt 30300 caggaagaga atatggcaag gacacgtggt cactgtataagggaggcaat ctaagatgtg 30360 tcctagaaac tggagaatgg gctacaaaga aagcagcactaaggaacaat gctgcagagg 30420 gaaactgttg cagaatattg agggtgtcag tgagtttgtatgtaactgca agcagagagt 30480 cactggaggt tgggtagtaa caaaatagga ggtggctcagacatttccac atgcaaatag 30540 attcagtagt tcctttttag ttcaaatgaa ccttctattgcccttattag tgattctata 30600 aagtaaaatc tacacagtgc agagggtggc cttagaggctaacgagcctg gtttcctgcc 30660 tcagctgccc tcactgagtg taggggtgct ttctttaatctctggaaacc tccactgtct 30720 catctgtaaa atggagataa tactaacacc ttgatgtgatgtcatgaaga gaaattaaga 30780 gaggcagtgt aagtaaagtc cccacataga gcctgggacatacaagccac ttcataagtg 30840 tcaattctta ttgaaccttt ttattaagaa actaacaatattctatcatt ctggacctac 30900 aaaagggcaa tttcatgtgg ctcaacttaa ggtttaggggaagcagtgag agaaatgaca 30960 acttgatgct tgtccattgt gacatgacag acctcttgacaagctaagac tcccattgtg 31020 atgagcctct cacacctggc cattccaatg gaacagacagggtaaggacc aatctggact 31080 gtgttatctt ttccaggtgc aagtatgtgc tatgggtaagtgccagtttg gagaactccc 31140 ttagccacag gaaatgaaaa ttcatgtgat tgtttgaaggattcagcttc tctttgctgc 31200 taatccttgg gttttgtgca cctagaatgt ggtctcctgcaggccctgaa agccttgaat 31260 tcctggcatc tttgctgtga aggtctccct ggctgctgctggaggaaggg gctggaagga 31320 gtgagtgtgt gcacaggttc agagttcagt cttcagacaaaaggagtgag ataaattgaa 31380 gacaagctgc cgatggtagt gcatggaact gctcaatgaccagcttcctt agcgaaaaca 31440 ttagcaacac attcaggcaa agggatgcga gaagttaagtactttgcaga aatatttgac 31500 aggccctgca aacactgagc aagaaaccat aggttctccccaattgcagg gatgcaagta 31560 acgtgaacac tttcctttcg gtcatcttcc ttggtggtcaggcatcatct ggatcacttt 31620 catctggcat cgggttataa ctacctgacc ctctcagactggggtgaatg tatcatcttt 31680 ccaaggtgtt tgccgttccc aacaaagaga ggaagccagttcgctattgg cctgttagct 31740 ttacaaacgg atggtagagc ttatgcttac caaggaagagtgaaagggga ttatcgacca 31800 cttgttgaca gggaaaatag tttaatcaaa ctgtaactcagctactcatg gccactgaga 31860 aatctgagaa agcctctgtc ataataacac acataataatcctagtatta gaaagccctg 31920 cgctctggct aagactctac tatacttttc agtaacttatttccccagaa tctccatagg 31980 gatgcaattc cttcacccct gctttaagtt acttctctctcctcgcctca gtgatgtcat 32040 catatacacc tgtggacaaa agccgtgaca gggaaggagatgccatttac gtccctggtg 32100 attctatagg aaactaaggg acctccttat cacccttctatgaactatgc ccctgtcagc 32160 tttaaaaatt tgttgttgtt attccaattt ttttttttttgagatggagt ttcactcttg 32220 ttgcccaggc tggagtgcaa tggcacaatc tcggctcaccacaacctctg cctcccaggt 32280 tcaaaccatt ctcctgcctc agcctcctga gtagctggaattacaggcat gcgccaccac 32340 gctcggctaa ttttgtattt ttagtggaaa cgggttttctccatgttgtt caggctggtc 32400 ttgaactcct gacctcaggt gatccgcccg cctcaggctcccaaagtgct gggattacag 32460 gcatgagcca gcacacctga cctgttattc caatttaacagttctttctt cccatacctg 32520 taaatgtgtg tgactgtgtg tgtgtgcatg tacactcacacacacacaaa tacacaagtt 32580 caagtgaaat ctaaatgctt ggtaaaacag tccatgtgcactaatttgca agagttgttg 32640 tgagggtaga gcttttgaat aaacataggt tgtcaaaggaaaaactccct ctgtgtaagc 32700 cacaggacaa aggttttgaa acacctttgt tatctaaagctggaaagaaa tgtcttgcct 32760 taaaagaatt tgcacattcg tacctctttc cacaaatacgtgaaaggacg tgcttttgaa 32820 gataagaaaa gtttaaattc tacaaaaaaa aaaaatctgatttgggcaga actcattgct 32880 cccttttctc tgtttctacc ttgttcttct ctgggtggatcatttactac ttatactgtc 32940 agtgttggtg ttgcttgttt acttagatcc ctgaagtcgagttgcacaac tccagggggc 33000 attcagataa aatatcatgt gaatgatgcc ctggagttttgcaggtagct ttgtcctgaa 33060 gacagggaca aaaatgtttc atctctttac ctcccagtgcctggtggaca tgccttcgga 33120 ccaagtagtt gcacaattca ttgttgctta gcaaatgaacaaatatgttc acctcactaa 33180 atagctgaca tgaaaacatt ttaaaaatag tatcaagatatttaaacagt cgattttatg 33240 aatttaaaag acacctagag atactacaat tccgtagttttttagattaa aaaacaaaga 33300 cccaaagtct atatttttta aaggaaggcc cagatggttttgtgaaggtc atgtgagtat 33360 ttagggacaa aactagagct gaaactcaat tctcttggccccaggtgatc ttctcactcc 33420 accagactta ctcagttcac atcacagtca caattcagattagagctatg aacaattcta 33480 tccatttgca caattctaac tggtgtttct aacttcattaaaagactctg aattattttt 33540 cttatatacc tctaatcaag atcatttggt attatcctgcatatgttcaa atgttaccta 33600 tctacagata tttgaactta ggtggggaat atctccacaaagtccattaa gtaagttcag 33660 ttttagtgaa aactgagatg gtgcagcttg agagattaagtgtagaattt ccaatgtaat 33720 gctttgaatg tgtaccttaa atctgtatca ctggcttattctgggaattg aagtcttatt 33780 tcatttctca gagaatgatg gttctgctac cagtaatctttaagggttag atcattcggg 33840 ttttttgttt gtttgagact aaataaatga agaaaacacatgttagatac aaaacactag 33900 aaatatatta attttcactg gagcgacacc aaaggccatcgacattaaaa atgaactcct 33960 aagttctttg caattcccca ggtatagatt taatatacaacacatgcatc tcttgaaact 34020 ctttctttgc tagtaagaat tattctcctg aaatacccacctgtcaaaaa gaaaggtaac 34080 attattgatt tttagaattc ttatttctgt cgtgtcagtaagcaataccg gaaagaaaat 34140 caaacactca ggagaattgg catgatggtg aaggttgagcttacaagtac agtggactca 34200 agtatccatg atccagcgca ctgagcaata aatccaaatgagcagtgacc acaggaaaac 34260 aatatgcagg gaggcctcgc tgggaaaagc taaacttttatatatgggaa tagtctatgg 34320 aggattacag gggatgtttt cttgggggta taaggtctggagtgtgcagt actgggtgaa 34380 gcccttatct aacaggcaac agaaaggtct tcccaggttaggcacacgtg actctacctc 34440 caacacagaa tttttttttt taagaaagca acagaaatttgcaaatgata gtctggtctt 34500 ttgtcctctc aattttaaag caaataacca gtattgtgttatctaccttt tcatggatgc 34560 atccagtgtg cctagaaggg ccagacttta ttctgtatttgcaacaaaag tagacccagc 34620 aactgatggg aagatatctg attgggaagc agaagcagctggtattttaa atcaggatga 34680 aagctaagat tttaggactc acttttgata ggaagaaaggatatatcaat tttcctttta 34740 atgagtggga tttttgaggt actttgtggg gcctctggttcaagactgtg gccagtgtgg 34800 tgttgtagga ggggcactga tggagagcta tcccggtgcattatttctga gccacctctg 34860 tgcactttac ttcctcattt gtaacatggg actaatgtgccctgctgagt cctcagagtt 34920 gctgcaacaa tcaaatgagc tattaaggga taagctcttttccagctacc tatgagaatg 34980 ggtatgatat ggtcccagaa tttgctctct agagccacagaaatctctta acccctacaa 35040 gaactcttta aagttgttat ccccattata tagatgaggaaactgagact tagacaaaaa 35100 gttgtccaag atcacataat attaaggaac agagctgggatgaatatttg agtctaattc 35160 caaaacattc tggaataact caatatgtgg ttttccatttctcccaaaaa caggtacctg 35220 cttttttcag tggcttgtgt tccagctgac agctccagggcctgtttgaa taattcgaag 35280 acaatcctta gttaggaaag caagctttaa ttatcactggggaacagaag gccgcatctt 35340 cgaggaattt ggcagacctc agcagggggc aaccacaggcctttggcaaa agatcacttt 35400 tcaacaacat tgtcaattcc agtgaccccc gaccttccacctgcaggtcc ctgaacagct 35460 gctgttctgt gggaagcagt ggcagtctgt cttcctttaaaaggcacatg cacactctgt 35520 ccctgctgcc tgctgagatc ccacctggga cctcatccccagagctgggg gtcatctccc 35580 atatcaagaa attaagaaaa ataagggggg gcaggaaaggacagctttga caacagtccc 35640 tgaactttcc cttttaatat aagccagatt taacgtatgtcattctgtaa atccgggagt 35700 ccaatttgag gcttgtaatt tgctgcaagc ttccctgttcctccaagtgg ggtggagcta 35760 tgccaagcac atcaaggtaa ctggtggaag atataatttccccactgtga gcctgcattt 35820 cagttcccta ttgtaatttt tatttgtgtt gaggttttgtggttttaaaa aagtcaacca 35880 gatttatttt taaattaacc cagcccaaca tcaaaggcaataagtagagg atgtttaggt 35940 attataaaga aaccctgtgt aatctgttat agctgtattctttctcaggg catgtaatgg 36000 taaatggtta ggggcctttc acaaccaact ttctatatttctctgacctc ggactaccct 36060 catgggcaaa aaaccctttt tgaggggatt tagtagcccctctctcctcc tcctcaacct 36120 cttaatctaa tcctgtttgt aacgcaacat gctgcatgaagaatacgaaa catgggctta 36180 agtccctccc cacttccctt atgactgtgg tttacttttagatatgaaga actctttcag 36240 gccaaaaaaa aaaaaagggg gggaccattt ggttaacgaaccattttctt tggtaggcag 36300 gagaaagttt atattgaaag tttatcttaa ggatgacaggtcatacctga agggtttgtt 36360 ttggaatact gtggattttt tctaacccaa ataattacaagagagttcct tgtttattgg 36420 ctcatggagg aaattcaagc gcctctgttc taggcattttaagtgctctg tatatatggt 36480 ggttgttcct caaaacagct ttgggtttgt tttttgttttttgttgttag tggtggtttt 36540 ttgagataga gtctcgctct gttacccagg ctggagtgcagtggcgcaat ctcggctcac 36600 tgcaccctcc acctctctgg ttcaaaagat tctcctgcctcagcctcctg agtagctggg 36660 attacaggcg cccgccacca tgcccagcta atttttgtatttttagtaga gacggggttt 36720 ccccatgttg gccaggctgg tctcgaactc ctgacctcaggtgattcacc cgcctgggcc 36780 ttccaaaatg ctgggattac aggcgtgagc caccatggctggcccaaaac agctttttaa 36840 gaaaagtgct attaacccca tttacagatg agcacatttgagccacaccc tcttttccac 36900 actctaaatc ttgtctcttt ctttaaacat gagttacttatacttctgag cataactgag 36960 gcacttttag agacagtgtc ttctaagctc aatgtgatattatttgtgct gctgtgctgc 37020 tactgggtaa ccagcaccca tcctggtcac cagggtaactttgtcaacca agaaggccaa 37080 ggatcccaaa ccagcatttt ctactatcaa aagagaggttctgcaaatcc actggcagga 37140 gagaaaatat aatagcaggt ggcatttata tgacccagtgtgcatggcag tgtcccaggg 37200 tatcaccgtg aatctcagaa actccaggct ttccccatgggaaatccaca ccaccacaga 37260 tccagtggag gactcggtca agactcctga aatcaaagaactcacagtga ctgattcttt 37320 ccctagtttt ataatataaa taatggcatg gggtcacattcagccgtcat tatccacatc 37380 atttcactga tgggatcctc ctcagacaga gattgggaatcagatttctc ggtcacataa 37440 actgttgctc attctgtgag gctgcctatt tgtaaagttgtggttcttat taaaagcaat 37500 ctcagacgta gcaagcaccc ctcacttccc ttctcattcattttgttaaa gcaaatgggc 37560 tttggaattc aggcctgttt ctaccactta ctaatgttgttaatttggag gagttcctca 37620 actttgccaa ggcttgattt tctctgctgt aaagagggaataataaacct attttacaga 37680 gcagctgaga caattaggtg agttaatgta tataaaatggtttgcataat acccaacaca 37740 tattaaactc tcactcggtt tttaatatta acctctatgtgcttaataac attgaagaag 37800 aagattcaag tagattatag tctgttaaag agttcaaatataaataaata attctcaggg 37860 tgagaattgc catagcatag atatgttacg tacccatggcagagcgtgag gtggcagcat 37920 ctaatggttg agggagttgg gtgagacatc aagagaaggtgacatatttt tttgagtacc 37980 cagtggaagg catgggatac catgtggatc tctgcagtagattaaatata gacttgaact 38040 aacctatcct ggaacaatag gacaatatcc ttgtggcttacagtaattat tccctgcacc 38100 tatattgatt tgtttattaa acgaatagct ttattggtaaacatgtatat tgcggaagta 38160 gacttggtta tcattcccac aagtccagtt aaagtaatggcatctatata aaaaactcat 38220 aaaaactaga tatgtaagta atcaataaaa tactcttctcaagtattcag gagaaaaaat 38280 gtgttgaaat gatgattcat cattccacat aacgtatttgtgactacatt taatagcctc 38340 attagcaata aaatttttat gagttaacat catatgagaatattcccttg taccttaccg 38400 agactttatc tgtagatttg taacataacc ataatcatcttggtatgttt cttacacatt 38460 ttattcagtg aacccaaatg aacttctaat tacatgttcagctgccagtc atggttttat 38520 atgtttgaat atatatacct tcagaggata tttgctctttggggtggtga agacttcatc 38580 ttcttataaa tgcaaacaga agatagttgg aagaggaaaatgttttagca gtgtctcaat 38640 tatctctcct taatgattat ttcacaacct cgagatattttcctaaaaga ctaagtaaga 38700 aatatatagt aagattcctt tctggatatt ttcagaactcctagttataa ctatgcacca 38760 aatatggata aacactggat tatgcagtac acaggaccaatgctgcccat ccacatggaa 38820 tttacaaaca ttctacagcg caaaaggctc cagactttgatgtcagtgga tgattctgtg 38880 gagagggtaa gcacatgaac ctacctcagt gatagtttttggcccagctt cctttgtgta 38940 gacttattct tgccaatcct gtttggtttt ttccccttcattttccagca tcattttgag 39000 agagaaagaa agagagagag tatgtgttta gtggcttaatcatccctccc ttatcttgtc 39060 ctcattccat ctacctctcc agggttggtt tcttatggagccagtaaaaa agaggagaga 39120 aaaatcaaat cagcgtagat caggggccac atcctcaaaggcaataaaga attgatggag 39180 cttgtgctga acttgaactt taagttaagg gccccatctaaaggaacagc aattactcag 39240 ctccagctaa attttgccat gtagaaatgt ggatcaagtatcagcaggtc ttctgacttt 39300 tttaaagaag ccagaaacac aaaaaatttt atttgaaattttctgaactt tgaaacatag 39360 tataagccaa acaagacgtg cctcaggctg gatttaactggatcaggctc agaagcagcc 39420 tgtttttaac ccttggtaat tagatatgtg atgataattttaacaatgga ttttcaaagt 39480 acaacctata aagtttgatg gtagaggttg ttgtgcggggtgtttttgtt tttgttctac 39540 caaacaagca aacaaaaagc ctaaaagtag aatgtgctagattccaaaaa gttacatttc 39600 acctttacca ttggaccttt ccctcccaga ctgtaagcaaatagaaaatg tggataatgt 39660 tattaaagca actcttgcct tttaaaaata acaggaaaaagatttggggg caatcgtggc 39720 aacactattg agcatcatct tataccagca caattgattctgacttgttc ctttgctgta 39780 tttcagctgt ataacatgct cgtggagacg ggggagctggagaatactta catcatttac 39840 accgccgacc atggttacca tattgggcag tttggactggtcaaggggaa atccatgcca 39900 tatgactttg atattcgtgt gccttttttt attcgtggtccaagtgtaga accaggatca 39960 atgtacgtat ttctctgttt gcaacattca actgtcgtacctcaagtgtg tctaagataa 40020 ttcaattacc agtctcagta tctggtttcc tttcatccaaaacaaaaaag gatgtgtgta 40080 ggctggttaa tttcgaagat gaaaaccttt tcctccctgccacatcttaa attagctcaa 40140 gtatactact taaagagaaa ggaaaaataa gtgtatcaatgactaattct ctcaaattga 40200 ctggaatcta tgtctttttg gtctgtgtgc acagacaggatgtgatcttc tgggatatca 40260 cccttctttg aatcagagat acgctgtcat ttaaaaaaaaaacctgacac catcctttta 40320 gtgtttaact tttaaaaatt attccgaaag aaatgtttttaaaagataaa ttttgaaaag 40380 ctggcttttc ttttaaagga aaaagagcta aaggactaggctgctatttc tgtcactgta 40440 ggcaggtcac tgcttctctt tgcatctcta ttttcccatcatgaaatggc cttgcctatt 40500 ttcccatcat aaaatggcct tgtcaatcat ctcaggatgttttgaataaa atgggattgc 40560 atccatgaaa gaattatgga aagactaaaa gaaaaagtggaagtagaatc cagaagctgg 40620 aatggccctt gaaaagcatc tagcctggac ccctgatgtaacagtgaagc taggcagaca 40680 gcgtcagtgc ccctctcaga tccctgattc tggaagtggtggcagtgggg cccagagccc 40740 agatgttctg aatctccccc gagtaccaca tgtgctgctgcatcatacta catcctggag 40800 agagggcaat tgaagtaaga aaggtttctt tgaagaatgttattgtgctt cctaagatta 40860 ttagaaacac cctgagaatt gtcagatcta tatctagaaggctttatgtt ttaaggacta 40920 gatagcatag attgaattca actgtaaaaa ctgtacatccttttttaaaa tattgatttt 40980 aaagctcttg ttcaacggaa aagttatgga ctattgggttttaagcaaaa gtatgcttgc 41040 tgcaaaaacc atgtatcagg ctgcatcttg cctggtgatgtggtcagaat acaggggtgc 41100 aggcatctct ccagcctgac cctggcaaga gtcagttaatcttgctcagt gccattgctg 41160 tgatcacaca cccacccttg ccacacaact acccatgcctaggagaccag atgagagggt 41220 gagaagagtt gaaggccaat gagtcactgc tgtagaaaaagcagccctaa gtgccacctt 41280 cccctggcat tggatctcag ccatcaccgt gtgcccctttacagagtccc acagatcgtt 41340 ctcaacattg acttggcccc cacgatcctg gatattgctgggctcgacac acctcctgat 41400 gtggacggca agtctgtcct caaacttctg gacccagaaaagccaggtaa caggtgtgtc 41460 attgttcctc ctctcagcca gccccaaata cactgagctccagctggtgc ccagagccag 41520 ccagcagctg aagacatgga ggcagaatat gccttgcccacaaggatcac cccaagctga 41580 gcatttctca gctgcttgtg aatagcatat tgatggagatgcactcatgg tctgtgggaa 41640 gtgagaggtg tttctttaaa taagctgtta gcacagatccatttggaaaa acgtccagat 41700 gccaaaagta aatattatca ttttgctttc aggtttcgaacaaacaagaa ggccaaaatt 41760 tggcgtgata cattcctagt ggaaagaggg taattattggttcctggggt gcttctggga 41820 accagtccta gtgggcagct ttccctgctg agtattttttttctccttat ttttgtttac 41880 taagcatgca gatttcgtaa acctagtcac aagattgaatggtttgctgc ttattctgta 41940 gtggtcaata gagtaataat tgctggatca gaattgtaaagaataaccct caagttggtt 42000 aattggtaca aaaacacagt tagatagaag ttatagaatttgatagtata gttgggacat 42060 tatcgttaac aataatttat gtatatctta aaatagctagaagtgaagaa ttgcaaagtt 42120 cccaacacaa ggaaaagata aatgagatga tgaatatcccaattatcttg atttgatcat 42180 tacacattgt agactggtat ccatatatca cacgtacccccaaaatatgt ataattgtga 42240 tatatcaatt tttaaaatac caaaaaagca agagaatgacgactccacat cccccaaaaa 42300 gaataaattc tcataagctt ggaccaaagc ctttatcatgggtgtagatt tactgttgca 42360 tttctcagtg ctggtttcta atcagaccag tggattgagtttctctacca tcctccccac 42420 gttcttctct aagctgcctc caagcctcac ccggcacccttcttcctact tcctacttct 42480 tttccttgtg tgcctttcct agttttaaat agataaatgtatgccattgt aattatttcc 42540 attgtcactt ctgggtttcc ccttttggtt c 42571 4360 PRT Human 4 Met Lys Tyr Ser Cys Cys Ala Leu Val Leu Ala Val Leu GlyThr Glu 1 5 10 15 Leu Leu Gly Ser Leu Cys Ser Thr Val Arg Ser Pro ArgPhe Arg Gly 20 25 30 Arg Ile Gln Gln Glu Arg Lys Asn Ile Arg Pro Asn IleIle Leu Val 35 40 45 Leu Thr Asp Asp Gln Asp Val Glu Leu Gly Ser Leu GlnVal Met Asn 50 55 60 Lys Thr Arg Lys Ile Met Glu His Gly Gly Ala Thr PheIle Asn Ala 65 70 75 80 Phe Val Thr Thr Pro Met Cys Cys Pro Ser Arg SerSer Met Leu Thr 85 90 95 Gly Lys Tyr Val His Asn His Asn Val Tyr Thr AsnAsn Glu Asn Cys 100 105 110 Ser Ser Pro Ser Trp Gln Ala Met His Glu ProArg Thr Phe Ala Val 115 120 125 Tyr Leu Asn Asn Thr Gly Tyr Arg Thr AlaPhe Phe Gly Lys Tyr Leu 130 135 140 Asn Glu Tyr Asn Gly Ser Tyr Ile ProPro Gly Trp Arg Glu Trp Leu 145 150 155 160 Gly Leu Ile Lys Asn Ser ArgPhe Tyr Asn Tyr Thr Val Cys Arg Asn 165 170 175 Gly Ile Lys Glu Lys HisGly Phe Asp Tyr Ala Lys Asp Tyr Phe Thr 180 185 190 Asp Leu Ile Thr AsnGlu Ser Ile Asn Tyr Phe Lys Met Ser Lys Arg 195 200 205 Met Tyr Pro HisArg Pro Val Met Met Val Ile Ser His Ala Ala Pro 210 215 220 His Gly ProGlu Asp Ser Ala Pro Gln Phe Ser Lys Leu Tyr Pro Asn 225 230 235 240 AlaSer Gln His Ile Thr Pro Ser Tyr Asn Tyr Ala Pro Asn Met Asp 245 250 255Lys His Trp Ile Met Gln Tyr Thr Gly Pro Met Leu Pro Ile His Met 260 265270 Glu Phe Thr Asn Ile Leu Gln Arg Lys Arg Leu Gln Thr Leu Met Ser 275280 285 Val Asp Asp Ser Val Glu Arg Leu Tyr Asn Met Leu Val Glu Thr Gly290 295 300 Glu Leu Glu Asn Thr Tyr Ile Ile Tyr Thr Ala Asp His Gly TyrHis 305 310 315 320 Ile Gly Gln Phe Gly Leu Val Lys Gly Lys Ser Met ProTyr Asp Phe 325 330 335 Asp Ile Arg Val Pro Phe Phe Ile Arg Gly Pro SerVal Glu Pro Gly 340 345 350 Ser Ile Val Pro Gln Ile Val Leu 355 360 5307 PRT Human 5 Asp Val Glu Leu Gly Ser Leu Gln Val Met Asn Lys Thr ArgLys Ile 1 5 10 15 Met Glu His Gly Gly Ala Thr Phe Ile Asn Ala Phe ValThr Thr Pro 20 25 30 Met Cys Cys Pro Ser Arg Ser Ser Met Leu Thr Gly LysTyr Val His 35 40 45 Asn His Asn Val Tyr Thr Asn Asn Glu Asn Cys Ser SerPro Ser Trp 50 55 60 Gln Ala Met His Glu Pro Arg Thr Phe Ala Val Tyr LeuAsn Asn Thr 65 70 75 80 Gly Tyr Arg Thr Ala Phe Phe Gly Lys Tyr Leu AsnGlu Tyr Asn Gly 85 90 95 Ser Tyr Ile Pro Pro Gly Trp Arg Glu Trp Leu GlyLeu Ile Lys Asn 100 105 110 Ser Arg Phe Tyr Asn Tyr Thr Val Cys Arg AsnGly Ile Lys Glu Lys 115 120 125 His Gly Phe Asp Tyr Ala Lys Asp Tyr PheThr Asp Leu Ile Thr Asn 130 135 140 Glu Ser Ile Asn Tyr Phe Lys Met SerLys Arg Met Tyr Pro His Arg 145 150 155 160 Pro Val Met Met Val Ile SerHis Ala Ala Pro His Gly Pro Glu Asp 165 170 175 Ser Ala Pro Gln Phe SerLys Leu Tyr Pro Asn Ala Ser Gln His Ile 180 185 190 Thr Pro Ser Tyr AsnTyr Ala Pro Asn Met Asp Lys His Trp Ile Met 195 200 205 Gln Tyr Thr GlyPro Met Leu Pro Ile His Met Glu Phe Thr Asn Ile 210 215 220 Leu Gln ArgLys Arg Leu Gln Thr Leu Met Ser Val Asp Asp Ser Val 225 230 235 240 GluArg Leu Tyr Asn Met Leu Val Glu Thr Gly Glu Leu Glu Asn Thr 245 250 255Tyr Ile Ile Tyr Thr Ala Asp His Gly Tyr His Ile Gly Gln Phe Gly 260 265270 Leu Val Lys Gly Lys Ser Met Pro Tyr Asp Phe Asp Ile Arg Val Pro 275280 285 Phe Phe Ile Arg Gly Pro Ser Val Glu Pro Gly Ser Ile Val Pro Gln290 295 300 Ile Val Leu 305 6 309 PRT Drosophila melanogaster 6 Arg ProAsn Ile Ile Leu Ile Leu Thr Asp Asp Gln Asp Val Glu Leu 1 5 10 15 GlySer Leu Asn Phe Met Pro Arg Thr Leu Arg Leu Leu Arg Asp Gly 20 25 30 GlyAla Glu Phe Arg His Ala Tyr Thr Thr Thr Pro Met Cys Cys Pro 35 40 45 AlaArg Ser Ser Leu Leu Thr Gly Met Tyr Val His Asn His Met Val 50 55 60 PheThr Asn Asn Asp Asn Cys Ser Ser Pro Gln Trp Gln Ala Thr His 65 70 75 80Glu Thr Arg Ser Tyr Ala Thr Tyr Leu Ser Asn Ala Gly Tyr Arg Thr 85 90 95Gly Tyr Phe Gly Lys Tyr Leu Asn Lys Tyr Asn Gly Ser Tyr Ile Pro 100 105110 Pro Gly Trp Arg Glu Trp Gly Gly Leu Ile Met Asn Ser Lys Tyr Tyr 115120 125 Asn Tyr Ser Ile Asn Leu Asn Gly Gln Lys Ile Lys His Gly Phe Asp130 135 140 Tyr Ala Lys Asp Tyr Tyr Pro Asp Leu Ile Ala Asn Asp Ser IleAla 145 150 155 160 Phe Leu Arg Ser Ser Lys Gln Gln Asn Gln Arg Lys ProVal Leu Leu 165 170 175 Thr Met Ser Phe Pro Ala Pro His Gly Pro Glu AspSer Ala Pro Gln 180 185 190 Tyr Ser His Leu Phe Phe Asn Val Thr Thr HisHis Thr Pro Ser Tyr 195 200 205 Asp His Ala Pro Asn Pro Asp Lys Gln TrpIle Leu Arg Val Thr Glu 210 215 220 Pro Met Gln Pro Val His Lys Arg PheThr Asn Leu Leu Met Thr Lys 225 230 235 240 Arg Leu Gln Thr Leu Gln SerVal Asp Val Ala Val Glu Arg Val Tyr 245 250 255 Asn Glu Leu Lys Glu LeuGly Glu Leu Asp Asn Thr Tyr Ile Val Tyr 260 265 270 Thr Ser Asp His GlyTyr His Leu Gly Gln Phe Gly Leu Ile Lys Gly 275 280 285 Lys Ser Phe ProPhe Glu Phe Asp Val Arg Val Pro Phe Leu Ile Arg 290 295 300 Gly Pro GlyIle Gln 305

That which is claimed is:
 1. An isolated peptide consisting of an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequenceshown in SEQ ID NO:2, wherein said fragment comprises at least 10contiguous amino acids.
 2. An isolated peptide comprising an amino acidsequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequenceshown in SEQ ID NO:2, wherein said fragment comprises at least 10contiguous amino acids.
 3. An isolated antibody that selectively bindsto a peptide of claim
 2. 4. An isolated nucleic acid molecule consistingof a nucleotide sequence selected from the group consisting of: (a) anucleotide sequence that encodes an amino acid sequence shown in SEQ IDNO:2; (b) a nucleotide sequence that encodes of an allelic variant of anamino acid sequence shown in SEQ ID NO:2, wherein said nucleotidesequence hybridizes under stringent conditions to the opposite strand ofa nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a nucleotidesequence that encodes an ortholog of an amino acid sequence shown in SEQID NO:2, wherein said nucleotide sequence hybridizes under stringentconditions to the opposite strand of a nucleic acid molecule shown inSEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment ofan amino acid sequence shown in SEQ ID NO:2, wherein said fragmentcomprises at least 10 contiguous amino acids; and (e) a nucleotidesequence that is the complement of a nucleotide sequence of (a)-(d). 5.An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence thatencodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotidesequence that encodes of an allelic variant of an amino acid sequenceshown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; (c) a nucleotide sequence that encodes anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3;(d) a nucleotide sequence that encodes a fragment of an amino acidsequence shown in SEQ ID NO:2, wherein said fragment comprises at least10 contiguous amino acids; and (e) a nucleotide sequence that is thecomplement of a nucleotide sequence of (a)-(d).
 6. A gene chipcomprising a nucleic acid molecule of claim
 5. 7. A transgenic non-humananimal comprising a nucleic acid molecule of claim
 5. 8. A nucleic acidvector comprising a nucleic acid molecule of claim
 5. 9. A host cellcontaining the vector of claim
 8. 10. A method for producing any of thepeptides of claim 1 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(d) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 11. A method for producing anyof the peptides of claim 2 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(d) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 12. A method for detecting thepresence of any of the peptides of claim 2 in a sample, said methodcomprising contacting said sample with a detection agent thatspecifically allows detection of the presence of the peptide in thesample and then detecting the presence of the peptide.
 13. A method fordetecting the presence of a nucleic acid molecule of claim 5 in asample, said method comprising contacting the sample with anoligonucleotide that hybridizes to said nucleic acid molecule understringent conditions and determining whether the oligonucleotide bindsto said nucleic acid molecule in the sample.
 14. A method foridentifying a modulator of a peptide of claim 2, said method comprisingcontacting said peptide with an agent and determining if said agent hasmodulated the function or activity of said peptide.
 15. The method ofclaim 14, wherein said agent is administered to a host cell comprisingan expression vector that expresses said peptide.
 16. A method foridentifying an agent that binds to any of the peptides of claim 2, saidmethod comprising contacting the peptide with an agent and assaying thecontacted mixture to determine whether a complex is formed with theagent bound to the peptide.
 17. A pharmaceutical composition comprisingan agent identified by the method of claim 16 and a pharmaceuticallyacceptable carrier therefor.
 18. A method for treating a disease orcondition mediated by a human enzyme protein, said method comprisingadministering to a patient a pharmaceutically effective amount of anagent identified by the method of claim
 16. 19. A method for identifyinga modulator of the expression of a peptide of claim 2, said methodcomprising contacting a cell expressing said peptide with an agent, anddetermining if said agent has modulated the expression of said peptide.20. An isolated human enzyme peptide having an amino acid sequence thatshares at least 70% homology with an amino acid sequence shown in SEQ IDNO:2.
 21. A peptide according to claim 20 that shares at least 90percent homology with an amino acid sequence shown in SEQ ID NO:2. 22.An isolated nucleic acid molecule encoding a human enzyme peptide, saidnucleic acid molecule sharing at least 80 percent homology with anucleic acid molecule shown in SEQ ID NOS :1 or
 3. 23. A nucleic acidmolecule according to claim 22 that shares at least 90 percent homologywith a nucleic acid molecule shown in SEQ ID NOS: 1 or 3.